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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

  • Current Issue
  • Advanced Online Publications Articles
  • Current Issue of Journal of Virology

    JVI Current Issue

  • Editorial Board [Masthead]

  • Recognizing the Top 25 Peer Reviewers for the Journal of Virology [Editorials]

  • Acknowledgment of Ad Hoc Reviewers [Editorials]

  • Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]

  • Evolution of Cocirculating Varicella-Zoster Virus Genotypes during a Chickenpox Outbreak in Guinea-Bissau [Genetic Diversity and Evolution]

  • Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history.

    IMPORTANCE VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations.

  • Latency-Associated Viral Interleukin-10 (IL-10) Encoded by Human Cytomegalovirus Modulates Cellular IL-10 and CCL8 Secretion during Latent Infection through Changes in the Cellular MicroRNA hsa-miR-92a [Cellular Response to Infection]

  • The UL111A gene of human cytomegalovirus encodes a viral homologue of the cellular immunomodulatory cytokine interleukin 10 (cIL-10), which, due to alternative splicing, results in expression of two isoforms designated LAcmvIL-10 (expressed during both lytic and latent infection) and cmvIL-10 (identified only during lytic infection). We have analyzed the functions of LAcmvIL-10 during latent infection of primary myeloid progenitor cells and found that LAcmvIL-10 is responsible, at least in part, for the known increase in secretion of cellular IL-10 and CCL8 in the secretomes of latently infected cells. This latency-associated increase in CCL8 expression results from a concomitant LAcmvIL-10-mediated suppression of the expression of the cellular microRNA (miRNA) hsa-miR-92a, which targets CCL8 directly. Taking the data together, we show that the previously observed downregulation of hsa-miR-92a and upregulation of CCL8 during HCMV latent infection of myeloid cells are intimately linked via the latency-associated expression of LAcmvIL-10.

    IMPORTANCE HCMV latency causes significant morbidity and mortality in immunocompromised individuals, yet HCMV is carried silently (latently) in 50 to 90% of the population. Understanding how HCMV maintains infection for the lifetime of an infected individual is critical for the treatment of immunocompromised individuals suffering with disease as a result of HCMV. In this study, we analyze one of the proteins that are expressed during the "latent" phase of HCMV, LAcmvIL-10, and find that the expression of the gene modulates the microenvironment of the infected cell, leading to evasion of the immune system.

  • HIV-1 Entry in SupT1-R5, CEM-ss, and Primary CD4+ T Cells Occurs at the Plasma Membrane and Does Not Require Endocytosis [Virus-Cell Interactions]

  • Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and coreceptor, leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry, and infection in T-cell lines and in primary CD4+ T cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the coreceptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) expression of dominant negative dynamin-2 was measured and was found to efficiently block HIV-1 endocytosis but to not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23ddeg;C, cells were incubated with HIV-1 at 22ddeg;C for various times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, entry at the plasma membrane was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after cells were shifted to 37ddeg;C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss, and primary CD4+ T cells, with little, if any, contribution coming from endocytosed virions.

    IMPORTANCE HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence regarding the site of HIV-1 fusion has been reported, with some studies claiming that fusion occurs predominantly at the plasma membrane, while others have suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4+ T cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4+ T cells.

  • Recent Population Expansions of Hepatitis B Virus in the United States [Genetic Diversity and Evolution]

  • The recent epidemic history of hepatitis B virus (HBV) infections in the United States is complex, as indicated by current disparity in HBV genotype distribution between acute and chronic hepatitis B cases and the rapid decline in hepatitis B incidence since the 1990s. We report temporal changes in the genetic composition of the HBV population using whole-genome sequences (n = 179) from acute hepatitis B cases (n = 1,206) identified through the Sentinel County Surveillance for Acute Hepatitis (1998 to 2006). HBV belonged mainly to subtypes A2 (75%) and D3 (18%), with times of their most recent common ancestors being 1979 and 1987, respectively. A2 underwent rapid population expansions in ca. 1995 and ca. 2002, coinciding with transient rises in acute hepatitis B notification rates among adults; D3 underwent expansion in ca. 1998. A2 strains from cases identified after 2002, compared to those before 2002, tended to cluster phylogenetically, indicating selective expansion of specific strains, and were significantly reduced in genetic diversity (P = 0.001) and frequency of drug resistance mutations (P = 0.001). The expansion of genetically close HBV A2 strains was associated with risk of infection among male homosexuals (P = 0.03). Incident HBV strains circulating in the United States were recent in origin and restricted in genetic diversity. Disparate transmission dynamics among phylogenetic lineages affected the genetic composition of HBV populations and their capacity to maintain drug resistance mutations. The tendency of selectively expanding HBV strains to be transmitted among male homosexuals highlights the need to improve hepatitis B vaccination coverage among at-risk adults.

    IMPORTANCE Hepatitis B virus (HBV) remains an important cause of acute and chronic liver disease globally and in the United States. Genetic analysis of HBV whole genomes from cases of acute hepatitis B identified from 1998 to 2006 in the United States showed dominance of genotype A2 (75%), followed by D3 (18%). Strains of both subtypes were recent in origin and underwent rapid population expansions from 1995 to 2000, indicating increase in transmission rate for certain HBV strains during a period of decline in the reported incidence of acute hepatitis B in the United States. HBV A2 strains from a particular cluster that experienced the most recent population expansion were more commonly detected among men who have sex with men. Vaccination needs to be stepped up to protect persons who remain at risk of HBV infection.

  • Evolution of Bluetongue Virus Serotype 1 in Northern Australia over 30 Years [Genetic Diversity and Evolution]

  • Bluetongue virus serotype 1 (BTV 1) was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long-term surveillance programs (1977 to 2011), 2,487 isolations of 10 BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1,019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In 3 years, no BTVs were isolated, and in 12 years, no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with 10 Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for coevolution of BTV genome segments. Evolutionary dynamics and selection pressure estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 x 10nndash;4 to 5.3 x 10nndash;4 substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection, with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate hosts) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses.

    IMPORTANCE Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints, with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

  • Activation of the RIG-I Pathway during Influenza Vaccination Enhances the Germinal Center Reaction, Promotes T Follicular Helper Cell Induction, and Provides a Dose-Sparing Effect and Protective Immunity [Vaccines and Antiviral Agents]

  • Pattern recognition receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type I interferons (IFNs). Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway for antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens coadministered with 5'ppp-double-stranded RNA (dsRNA), a RIG-I ligand, developed robust levels of hemagglutination-inhibiting antibodies, enhanced germinal center reaction, and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in the draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of the RIG-I pathway was able to reduce the antigen requirement by 10- to 100-fold in inducing optimal influenza-specific cellular and humoral responses, including protective immunity. The effects induced by 5'ppp-dsRNA were significantly dependent on type I IFN and IPS-1 (an adapter protein downstream of the RIG-I pathway) signaling but were independent of the MyD88- and TLR3-mediated pathways. Our results show that activation of the RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses, and this indicates the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines.

    IMPORTANCE The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate antiviral cytokines that help control viral replication and dissemination. In this study, we show that the RLR pathway can be effectively exploited to enhance adaptive immunity and protective immune memory against viral infection. Our results show that activation of the RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at low vaccine antigen doses. These results highlight the potential utility of exploiting the RIG-I pathway to enhance viral-vaccine-specific immunity and have broader implications for designing better vaccines in general.

  • Hyperglycosylated Stable Core Immunogens Designed To Present the CD4 Binding Site Are Preferentially Recognized by Broadly Neutralizing Antibodies [Vaccines and Antiviral Agents]

  • The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4+ CCR5+ host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy.

    IMPORTANCE Major obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified gp120 core.

  • Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid [Structure and Assembly]

  • Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed.

    IMPORTANCE The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

  • B Cell Response to Herpesvirus Infection of the Olfactory Neuroepithelium [Pathogenesis and Immunity]

  • Viruses commonly infect the respiratory tract. Analyses of host defense have focused on the lungs and the respiratory epithelium. Spontaneously inhaled murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1) instead infect the olfactory epithelium, where neuronal cilia are exposed to environmental antigens and provide a route across the epithelial mucus. We used MuHV-4 to define how B cells respond to virus replication in this less well-characterized site. Olfactory infection elicited generally weaker acute responses than lung infection, particularly in the spleen, reflecting slower viral replication and spread. Few virus-specific antibody-forming cells (AFCs) were found in the nasal-associated lymphoid tissue (NALT), a prominent response site for respiratory epithelial infection. Instead, they appeared first in the superficial cervical lymph nodes. The focus of the AFC response then moved to the spleen, matching the geography of virus dissemination. Little virus-specific IgA response was detected until later in the bone marrow. Neuroepithelial HSV-1 infection also elicited no significant AFC response in the NALT and a weak IgA response. Thus, olfactory herpesvirus infection differed immunologically from an infection of the adjacent respiratory epithelium. Poor IgA induction may help herpesviruses to transmit via long-term mucosal shedding.

    IMPORTANCE Herpesviruses are widespread, persistent pathogens against which vaccines have had limited success. We need to understand better how they interact with host immunity. MuHV-4 and HSV-1 inhaled by alert mice infect the olfactory neuroepithelium, suggesting that this is a natural entry route. Its immunology is almost completely unknown. The antibody response to neuroepithelial herpesvirus infection started in the cervical lymph nodes, and unlike respiratory influenza virus infection, did not significantly involve the nasal-associated lymphoid tissue. MuHV-4 and HSV-1 infections also elicited little virus-specific IgA. Therefore, vaccine-induced IgA might provide a defense that herpesviruses are ill-equipped to meet.

  • Type I Interferon Signaling Enhances CD8+ T Cell Effector Function and Differentiation during Murine Gammaherpesvirus 68 Infection [Pathogenesis and Immunity]

  • CD8+ T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons (IFNs) have been shown to have direct and indirect roles in supporting CD8+ T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1nndash;/nndash; mice) have high levels of reactivation during infection with murine gammaherpesvirus 68 (MHV68), a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8+ T cell response. To test this, IFNAR1nndash;/nndash; mice were infected with MHV68 and the CD8+ T cell response was analyzed. In the absence of type I IFN signaling, there was a marked increase in short-lived effector CD8+ T cells, and MHV68-specific CD8+ T cells had upregulated expression of PD-1 and reduced tumor necrosis factor alpha (TNF-aalpha;), gamma IFN (IFN-), and interleukin-2 (IL-2) production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8+ T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1nndash;/nndash; mice failed to improve CD8+ T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8+ T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8+ T cell response.

    IMPORTANCE The murine gammaherpesvirus MHV68 has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV), which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8+ T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8+ T cell response in other virus models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8+ T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8+ T cell function during gammaherpesvirus infection, potentially through suppression of MHV68 replication.

  • Modeling the Timing of Antilatency Drug Administration during HIV Treatment [Vaccines and Antiviral Agents]

  • Latently infected cells are considered a major barrier to the cure of HIV infection, since they are long-lived under antiretroviral therapy (ART) and cause viral replication to restart soon after stopping ART. In the last decade, different types of antilatency drugs have been explored with the aim of reactivating and purging this latent reservoir and the hope of achieving a cure. Because of toxicity and safety considerations, antilatency drugs can only be given for a short time to patients on long-term ART, with little effect. We recently investigated the turnover of latently infected cells during active infection and have found that it was strongly correlated with viral load. This implies that although latently infected cells had long life spans in a setting of a low viral load (such as during ART), they turned over quickly under a high viral load. Possible reasons for this could be that an increased viral load causes increased activation or death of CD4+ T cells, including those that are latently infected. Taking these results into account, we developed a mathematical model to study the most appropriate timing of antilatency drugs in relationship to the initiation of ART. We found that the best timing of a short-term antilatency drug would be the start of ART, when viral load, CD4+ T cell activation, and latent cell turnover are all high. These results have important implications for the design of HIV cure-related clinical trials.

    IMPORTANCE The antiretroviral therapy (ART) of HIV-infected patients currently needs to be lifelong, because the cells latently infected with HIV start new rounds of infection as soon as the treatment is stopped. In the last decade, a number of different types of antilatency drugs have been explored with the aim of "reactivating" and "purging" this latent reservoir and thus achieving a cure. These drugs have thus far been tested on patients only after long-term ART and have demonstrated little or no effect. We use mathematical modeling to show that the most efficacious timing of a short-term antilatency treatment may be the start of ART because of possible interactions of antilatency drugs with natural activation pathways.

  • Drosophila C Virus Systemic Infection Leads to Intestinal Obstruction [Pathogenesis and Immunity]

  • Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host defense in flies, which involves both RNA interference and inducible responses. Although lethality is used routinely as a readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies still are poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are expressed specifically in the midgut and also are repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila which results from intestinal obstruction with the accumulation of peritrophic matrix at the entry of the midgut and the accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of dipteran insects, the crop.

    IMPORTANCE DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies and show that it results from the tropism of the virus for an essential but poorly characterized organ in the digestive tract, the crop. Our results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.

  • Quantitation of Replication-Competent HIV-1 in Populations of Resting CD4+ T Cells [Pathogenesis and Immunity]

  • Central memory (TCM) CD4+ T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 17 patients and replication-competent HIV in 4 patients, pools of resting and activated transitional memory (TTM) CD4+ T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting TTM cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting-dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment but was infrequently detected within the resting TTM compartment. These observations suggest that prolonged ART may limit persistent latent infection in the TTM compartment. Our results confirm the importance of latent infection within the TCM compartment and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART or were treated very early in infection.

    IMPORTANCE Antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality among HIV-infected patients. However, HIV integrates into the genome of CD4+ T cells, generating pools of long-lived cells that are reservoirs of latent HIV. Two main subsets of CD4+ T cells, central memory and transitional memory cells, were reported to be major reservoirs of HIV infection. However, this study primarily measured the HIV DNA content, which also includes defective proviruses that would not be able to replicate and initiate new rounds of infection. By analyzing the replication-competent virus in both cell subsets, we showed that transitional memory cells may not be a durable reservoir in patients on successful ART.

  • DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity [Virus-Cell Interactions]

  • The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed of the PB1, PB2, and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nuclei of infected cells. In this study, we newly identified host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with the bPB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, while the J domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to enter the nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner.

    IMPORTANCE The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaptation. In this study, we newly identified a host protein, DnaJA1/Hsp40, that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nuclei of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into the nucleus along with the nuclear import of the PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, which is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of the Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs.

  • Suppressors of Cytokine Signaling 1 and 3 Are Upregulated in Brain Resident Cells in Response to Virus-Induced Inflammation of the Central Nervous System via at Least Two Distinctive Pathways [Pathogenesis and Immunity]

  • Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS upregulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection. In this study, we used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected central nervous system (CNS), and by employing various knockout mouse models, we sought to identify regulatory mechanisms that may underlie a virus induced upregulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual upregulation of SOCS1/3 mRNA expression peaking at day 7 postinfection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells, including astrocytes and some neurons, and for SOCS1 in particular this upregulation was almost entirely mediated by gamma interferon (IFN-) produced by infiltrating T cells. After infection with YF, we also found SOCS expression to be upregulated in brain resident cells with a peak on day 7 p.i., but in this model, the upregulation was only partially dependent on IFN- and T cells, indicating that at least one other mediator was involved in the upregulation of SOCS following YF infection. We conclude that virus-induced inflammation of the CNS is associated with upregulation of SOCS1/3 mRNA expression in brain resident cells and that at least two distinctive pathways can lead to this upregulation.

    IMPORTANCE In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We found that both a noncytolytic and a cytolytic virus induce marked upregulation of SOCS1 and -3 expression. Notably, the kinetics of the observed upregulation follows that of activity within proinflammatory signaling pathways and, interestingly, type II interferon (IFN), which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counterinflammatory response. Another key observation is that not only cells of the immune system but also CNS resident cells are actively involved in both the pro- and the counterinflammatory immune circuits; thus, for example, astrocytes upregulate both C-X-C-motif chemokine 10 (CXCL10) and SOCS when exposed to type II IFN in vivo.

  • Structurally Similar Woodchuck and Human Hepadnavirus Core Proteins Have Distinctly Different Temperature Dependences of Assembly [Structure and Assembly]

  • Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23ddeg;C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 mmu;M and 43.3 mmu;M, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ~5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4ddeg;C to 37ddeg;C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host.

    IMPORTANCE In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

  • A Combination of HA and PA Mutations Enhances Virulence in a Mouse-Adapted H6N6 Influenza A Virus [Pathogenesis and Immunity]

  • H6N6 viruses are commonly isolated from domestic ducks, and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity toward humans is not known. To address this, we generated a mouse-adapted (MA) swine influenza H6N6 virus (A/swine/Guangdong/K6/2010 [GDK6-MA]) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M), and hemagglutinin ([HA] L111F, H156N, and S263R) occurred in GDK6-MA. HA with the H156N mutation [HA(H156N)] resulted in enlarged plaque sizes on MDCK cells and enhanced early-stage viral replication in mammalian cells. PA(I38M) raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence; however, a combination of HA(H156N S263R) with PA(I38M) in the GDK6 backbone led to a significantly more virulent variant. This suggests that these substitutions can compensate for the lack of PB2(627K) and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health.

    IMPORTANCE Avian H6N6 influenza viruses are enzootic in domestic ducks and have been detected in swine in China. Infections of mammals by H6N6 viruses raise the possibility of viral adaptation and increasing pathogenicity in the new hosts. To examine the molecular mechanisms of adaptation, a mouse-adapted avian-origin swine influenza H6N6 virus (GDK6-MA), which had higher virulence than its parental virus, was generated. Specific mutations were found in PB2 (E627K), PA (I38M), and HA (L111F, H156N, and S263R) and were assessed for their virulence in mice. The combination of HA(H156N S263R) and PA(I38M) compensated for the lack of PB2(627K) and showed increased pathogenicity in mice, revealing a novel mechanism that can affect the virulence of influenza viruses. H6N6 viruses should be monitored in the field for more virulent forms that could threaten human health.

  • Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans [Genome Replication and Regulation of Viral Gene Expression]

  • The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3' part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2nndash;Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection.

    IMPORTANCE This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2nndash;adenovirus coinfection. This study shows that the two inhibitory processes are very different with regard to AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3' nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV ITRs. These findings have important implications both for a basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

  • Role of the Short Telomeric Repeat Region in Marek's Disease Virus Replication, Genomic Integration, and Lymphomagenesis [Transformation and Oncogenesis]

  • Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that causes generalized polyneuritis and T-cell lymphomas in chickens. MDV is able to integrate its genome into host telomeres, but the mechanism of integration is poorly understood. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome: multiple telomeric repeats (mTMR), with a variable number of up to 100 repeats, and short telomeric repeats (sTMR), with a fixed number of 6 repeats. The mTMR have recently been shown to play an important role in MDV integration and tumor formation; however, the functions of the sTMR have remained unknown. In this study, we demonstrate that deletion of the sTMR in the MDV genome abrogates virus replication, while extensive mutation of the sTMR does not, indicating that the presence of the sTMR but not the sTMR sequence itself is important. Furthermore, we generated a panel of truncation mutants to determine the minimal length of the sTMR and observed a direct correlation between sTMR length and MDV replication. To address the role of sTMR in MDV replication, integration, and tumorigenesis, sTMR sequences were replaced by a scrambled repeated sequence (vsTMR_mut). vsTMR_mut replicated comparably to parental and revertant viruses in vitro. In vivo, however, a significant reduction in disease and tumor incidence was observed in chickens infected with vsTMR_mut that also correlated with a reduced number of viral integration sites in tumor cells. Taken together, our data demonstrate that the sTMR play a central role in MDV genome replication, pathogenesis, and MDV-induced tumor formation.

    IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes high economic losses in the poultry industry. MDV integrates its genetic material into host telomeres, a process that is crucial for efficient tumor formation. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome that are identical to host telomeres and that are termed mTMR and sTMR. mTMR have been recently shown to be involved in MDV integration, while the functions of sTMR remain unknown. Here, we demonstrate that the presence and length of sTMR sequence, but not the exact nucleotide sequence, are crucial for MDV replication. Furthermore, the sTMR contribute to the high integration frequency of MDV and are important for MDV pathogenesis and tumor formation. As a number of herpesviruses harbor arrays of telomeric repeats (TMR), MDV serves as a model to determine the role of the herpesvirus TMR in replication, integration, and pathogenesis.

  • Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles [Structure and Assembly]

  • The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-AAring; cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly.

    IMPORTANCE Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

  • Different Roles of the Three Loops Forming the Adhesive Interface of Nectin-4 in Measles Virus Binding and Cell Entry, Nectin-4 Homodimerization, and Heterodimerization with Nectin-1 [Virus-Cell Interactions]

  • Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies viruses, use ubiquitous nectins 1 and 2. The negative-strand RNA virus measles virus (MeV) uses tissue-specific nectin-4, and the positive-strand RNA virus poliovirus uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C'C'', and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C'C'' loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C'C'' loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation and MeV-induced fusion.

    IMPORTANCE Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin requires only the BC and FG loops. However, we discovered that residues in the C'C'' loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

  • Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons [Virus-Cell Interactions]

  • Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 mmu;m/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 mmu;m/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 mmu;m/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons.

    IMPORTANCE Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

  • RNA Populations in Immunocompromised Patients as Reservoirs for Novel Norovirus Variants [Genetic Diversity and Evolution]

  • Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4). Novel GII.4 strains emerge every 2 to 4 years and replace older variants as the dominant norovirus. Novel variants emerge through a combination of recombination, genetic drift, and selection driven by population immunity, but the exact mechanism of how or where is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full-genome bioinformatic analysis of publicly available GII.4 norovirus sequences from 1974 to 2014 to identify converging sites. Variable sites were proportionally more likely to be within two amino acids (P llt; 0.05) of positively selected sites. Further analysis using a hypergeometric distribution indicated that polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites.In conclusion, random mutations may have a positive impact on driving norovirus evolution, and immunocompromised individuals could serve as potential reservoirs for novel GII.4 strains.

    IMPORTANCE Norovirus is the most common cause of viral gastroenteritis in the United States. Every 2 to 3 years novel norovirus variants emerge and replace dominant strains. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study, we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatic analysis, we showed that most genetic polymorphisms in the novel variants occur near 0 to 2 amino acids of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size and number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants, and although mutations in viruses are random, they can have a positive effect on viral evolution.

  • Nipah Virion Entry Kinetics, Composition, and Conformational Changes Determined by Enzymatic Virus-Like Particles and New Flow Virometry Tools [Virus-Cell Interactions]

  • Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a bbeta;-lactamase (bbeta;La) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the bbeta;La fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses.

    IMPORTANCE Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. For example, drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses.

  • Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism [Virus-Cell Interactions]

  • Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types.

    IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression.

  • The 2'-5'-Oligoadenylate Synthetase 3 Enzyme Potently Synthesizes the 2'-5'-Oligoadenylates Required for RNase L Activation [Cellular Response to Infection]

  • The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS.

    IMPORTANCE We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

  • Vaccine-Induced CD107a+ CD4+ T Cells Are Resistant to Depletion following AIDS Virus Infection [Vaccines and Antiviral Agents]

  • CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction following virus infection. However, virus-specific CD4+ T cells can be preferential targets for human immunodeficiency virus (HIV) infection. HIV-specific CD4+ T-cell induction by vaccination may thus result in enhancement of virus replication following infection. In the present study, we show that vaccine-elicited CD4+ T cells expressing CD107a are relatively resistant to depletion in a macaque AIDS model. Comparison of virus-specific CD107a, macrophage inflammatory protein-1bbeta;, gamma interferon, tumor necrosis factor alpha, and interleukin-2 responses in CD4+ T cells of vaccinated macaques prechallenge and 1 week postchallenge showed a significant reduction in the CD107anndash; but not the CD107a+ subset after virus exposure. Those vaccinees that failed to control viremia showed a more marked reduction and exhibited significantly higher viral loads at week 1 than unvaccinated animals. Our results indicate that vaccine-induced CD107anndash; CD4+ T cells are depleted following virus infection, suggesting a rationale for avoiding virus-specific CD107anndash; CD4+ T-cell induction in HIV vaccine design.

    IMPORTANCE Induction of effective antibody and/or CD8+ T-cell responses is a principal vaccine strategy against human immunodeficiency virus (HIV) infection. CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction. However, virus-specific CD4+ T cells can be preferential targets for HIV infection. Here, we show that vaccine-induced virus-specific CD107anndash; CD4+ T cells are largely depleted following infection in a macaque AIDS model. While CD4+ T-cell responses are important in viral control, our results indicate that virus-specific CD107anndash; CD4+ T-cell induction by vaccination may not lead to efficient CD4+ T-cell responses following infection but rather be detrimental and accelerate viral replication in the acute phase. This suggests that HIV vaccine design should avoid virus-specific CD107anndash; CD4+ T-cell induction. Conversely, this study found that vaccine-induced CD107a+ CD4+ T cells are relatively resistant to depletion following virus challenge, implying that induction of these cells may be an alternative approach toward HIV control.

  • HIV-1 Nef and Vpu Are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors, Including Tetraspanins [Virus-Cell Interactions]

  • HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share specificity for some molecules such as CD4 and major histocompatibility complex class I (MHC-I), while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu, or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptor levels on A3.01 T lymphocytes. Thirty-six out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for the spread of HIV-1 in the infected host.

    IMPORTANCE In this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the coevolution of Nef and Vpu function.

  • HIV-1 Envelope Glycan Moieties Modulate HIV-1 Transmission [Virus-Cell Interactions]

  • The HIV-1 envelope protein (Env) is heavily glycosylated, with approximately 50% of the Env molecular mass being contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (i) transcytosed across an epithelial cell monolayer, (ii) attached to monocyte-derived macrophages (MDMs), (iii) bound monocyte-derived dendritic cells (MoDCs), and (iv) trans-infected primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes, and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing a crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission.

    IMPORTANCE HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In the study described in this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

  • Hijacking Dlg1 for Oncogenic Phosphatidylinositol 3-Kinase Activation in Human Epithelial Cells Is a Conserved Mechanism of Human Adenovirus E4-ORF1 Proteins [Transformation and Oncogenesis]

  • The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes viral replication as well as cellular metabolic reprogramming, survival, and transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). We recently reported that the E4-ORF1 protein from subgroup D human adenovirus type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving separate interactions of E4-ORF1 with cellular discs large 1 (Dlg1) and PI3K to form a ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate whether other human adenovirus E4-ORF1 proteins share this mechanism of action. The results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 proteins encoded by representative human adenovirus serotypes from subgroups A to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular transformation. The first three of these E4-ORF1 activities were also observed in MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a conserved molecular mechanism of PI3K activation, which confers a common capacity to promote oncogenic transformation in human epithelial cells.

    IMPORTANCE PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular transformation by human adenovirus type 9, augments viral protein expression and replication by human adenovirus type 5, and dysregulates cellular glucose and lipid metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular mechanism to mediate constitutive PI3K activation that can provoke oncogenic transformation in human epithelial cells. The results raise potential safety concerns about the use of vectors encoding the E4-ORF1 gene in human gene therapy and vaccination. Our findings further suggest that the conserved mechanism revealed here may be targeted for development of therapeutic drugs to treat and prevent adenovirus-associated human diseases.

  • Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization [Vaccines and Antiviral Agents]

  • Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization.

    IMPORTANCE An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4+ and CD8+ T cell responses.

  • The Human Cytomegalovirus UL26 Protein Antagonizes NF-{kappa}B Activation [Virus-Cell Interactions]

  • Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NF-B pathway is a critical component that governs this response. We have found that the human cytomegalovirus (HCMV) UL26 protein antagonizes NF-B activation. Upon infection, an HCMV strain lacking the UL26 gene (UL26) induced the nuclear translocation of the NF-B RelB subunit and activated expression and secretion of interleukin-6 (IL-6), an NF-B target gene. The UL26 mutant was also more sensitive to challenge with tumor necrosis factor alpha (TNF-aalpha;), a canonical NF-B inducer. Further, expression of UL26 in the absence of other viral proteins blocked NF-B activation induced by either TNF-aalpha; treatment or infection with Sendai virus (SeV). Our results indicate that UL26 expression is sufficient to block TNF-aalpha;-induced NF-B nuclear translocation and IB degradation. Last, UL26 blocks TNF-aalpha;-induced IB-kinase (IKK) phosphorylation, a key step in NF-B activation. Combined, our results indicate that UL26 is part of a viral program to antagonize innate immunity through modulation of NF-B signaling.

    IMPORTANCE The NF-B signaling pathway regulates innate immunity, an integral host process that limits viral pathogenesis. Viruses have evolved mechanisms to modulate NF-B signaling to ensure their replication. HCMV is a major cause of birth defects and disease in immunosuppressed populations. HCMV is known to actively target the NF-B pathway, which is important for HCMV infection. Our results indicate that the HCMV UL26 gene is a key modulator of NF-B pathway activity. We find the UL26 gene is both necessary and sufficient to block NF-B activation upon challenge with antiviral cytokines. Further, UL26 attenuates the phosphorylation and activation of a key NF-B activating kinase complex, IKK. Our study provides new insight into how HCMV targets the NF-B pathway. Given its importance to viral infection, the mechanisms through which viruses target the NF-B pathway highlight areas of vulnerability that could be therapeutically targeted to attenuate viral replication.

  • Kaposi's Sarcoma-Associated Herpesvirus Downregulates Transforming Growth Factor {beta}2 To Promote Enhanced Stability of Capillary-Like Tube Formation [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), the most common tumor of AIDS patients worldwide. A key characteristic of KS tumors is extremely high levels of vascular slits and extravasated red blood cells, making neoangiogenesis a key component of the tumor. The main KS tumor cell is the spindle cell, a cell of endothelial origin that maintains KSHV predominantly in the latent state. In cultured endothelial cells, latent KSHV infection induces angiogenic phenotypes, including longer-term stabilization of capillary-like tube formation in Matrigel, a basement membrane matrix. The present studies show that KSHV infection of endothelial cells strongly downregulates transforming growth factor bbeta;2 (TGF-bbeta;2). This downregulation allows the stabilization of capillary-like tube formation during latent infection, as the addition of exogenous TGF-bbeta;2 inhibits the KSHV-induced stability of these structures. While two KSHV microRNAs are sufficient to downregulate TGF-bbeta;2 in endothelial cells, they are not required during KSHV infection. However, activation of the gp130 cell surface receptor is both necessary and sufficient for downregulation of TGF-bbeta;2 in KSHV-infected cells.

    IMPORTANCE Kaposi's sarcoma is a highly vascularized, endothelial cell-based tumor supporting large amounts of angiogenesis. There is evidence that KSHV, the etiologic agent of KS, induces aberrant angiogenesis. For example, KSHV induces stabilization of capillary-like tube formation in cultured endothelial cells. A clearer understanding of how KSHV regulates angiogenesis could provide potential therapeutic targets for KS. We found that KSHV downregulates TGF-bbeta;2, a cytokine related to TGF-bbeta;1 that is known to inhibit angiogenesis. The downregulation of this inhibitor promotes the stability of capillary-like tube formation insofar as adding back TGF-bbeta;2 to infected cells blocks KSHV-induced long-term tubule stability. Therefore, KSHV downregulation of TGF-bbeta;2 may increase aberrant vascularization in KS tumors through increased capillary formation and thereby aid in KS tumor promotion.

  • Linking Pig-Tailed Macaque Major Histocompatibility Complex Class I Haplotypes and Cytotoxic T Lymphocyte Escape Mutations in Simian Immunodeficiency Virus Infection [Pathogenesis and Immunity]

  • The influence of major histocompatibility complex class I (MHC-I) alleles on human immunodeficiency virus (HIV) diversity in humans has been well characterized at the population level. MHC-I alleles likely affect viral diversity in the simian immunodeficiency virus (SIV)-infected pig-tailed macaque (Macaca nemestrina) model, but this is poorly characterized. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIVmac251 genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIVmac251 at 4 to 10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms at both individual mutations and groups of mutations spanning 10-amino-acid segments were linked using in-house bioinformatics pipelines, since cytotoxic T lymphocyte (CTL) escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays, we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described to be CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and strategies for treatment of HIV infection.

    IMPORTANCE Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV, and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV type 1-infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing nearly identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaques to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques, and this enables a more refined level of analysis of future vaccine design and strategies for treatment of HIV infection.

  • Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of Its Effect on Virus-Specific CD8 T Cells [Virus-Cell Interactions]

  • The human cytomegalovirus (CMV) UL11 open reading frame (ORF) encodes a putative type I transmembrane glycoprotein which displays remarkable amino acid sequence variability among different CMV isolates, suggesting that it represents an important virulence factor. In a previous study, we have shown that UL11 can interact with the cellular receptor tyrosine phosphatase CD45, which has a central role for signal transduction in T cells, and treatment of T cells with large amounts of a soluble UL11 protein inhibited their proliferation. In order to analyze UL11 expression in CMV-infected cells, we constructed CMV recombinants whose genomes either encode tagged UL11 versions or carry a stop mutation in the UL11 ORF. Moreover, we examined whether UL11 affects the function of virus-specific cytotoxic T lymphocytes (CTLs). We found that the UL11 ORF gives rise to several proteins due to both posttranslational modification and alternative translation initiation sites. Biotin labeling of surface proteins on infected cells indicated that only highly glycosylated UL11 forms are present at the plasma membrane, whereas less glycosylated UL11 forms were found in the endoplasmic reticulum. We did not find evidence of UL11 cleavage or secretion of a soluble UL11 version. Cocultivation of CTLs recognizing different CMV epitopes with fibroblasts infected with a UL11 deletion mutant or the parental strain revealed that under the conditions applied UL11 did not influence the activation of CMV-specific CD8 T cells. For further studies, we propose to investigate the interaction of UL11 with CD45 and the functional consequences in other immune cells expressing CD45.

    IMPORTANCE Human cytomegalovirus (CMV) belongs to those viruses that extensively interfere with the host immune response, yet the precise function of many putative immunomodulatory CMV proteins remains elusive. Previously, we have shown that the CMV UL11 protein interacts with the leukocyte common antigen CD45, a cellular receptor tyrosine phosphatase with a central role for signal transduction in T cells. Here, we examined the proteins expressed by the UL11 gene in CMV-infected cells and found that at least one form of UL11 is present at the cell surface, enabling it to interact with CD45 on immune cells. Surprisingly, CMV-expressed UL11 did not affect the activity of virus-specific CD8 T cells. This finding warrants investigation of the impact of UL11 on CD45 functions in other leukocyte subpopulations.

  • Genome Analysis of the First Marseilleviridae Representative from Australia Indicates that Most of Its Genes Contribute to Virus Fitness [Genetic Diversity and Evolution]

  • The family Marseilleviridae consists of Acanthamoeba-infecting large DNA viruses with icosahedral particles ~0.2 mmu;m in diameter and genome sizes in the 346- to 380-kb range. Since the isolation of Marseillevirus from a cooling tower in Paris (France) in 2009, the family Marseilleviridae has expanded rapidly, with representatives from Europe and Africa. Five members have been fully sequenced that are distributed among 3 emerging Marseilleviridae lineages. One comprises Marseillevirus and Cannes 8 virus, another one includes Insectomime virus and Tunisvirus, and the third one corresponds to the more distant Lausannevirus. We now report the genomic characterization of Melbournevirus, the first representative of the Marseilleviridae isolated from a freshwater pond in Melbourne, Australia. Despite the large distance separating this sampling point from France, Melbournevirus is remarkably similar to Cannes 8 virus and Marseillevirus, with most orthologous genes exhibiting more than 98% identical nucleotide sequences. We took advantage of this optimal evolutionary distance to evaluate the selection pressure, expressed as the ratio of nonsynonymous to synonymous mutations for various categories of genes. This ratio was found to be less than 1 for all of them, including those shared solely by the closest Melbournevirus and Cannes 8 virus isolates and absent from Lausannevirus. This suggests that most of the 403 protein-coding genes composing the large Melbournevirus genome are under negative/purifying selection and must thus significantly contribute to virus fitness. This conclusion contrasts with the more common view that many of the genes of the usually more diverse large DNA viruses might be (almost) dispensable.

    IMPORTANCE A pervasive view is that viruses are fast-evolving parasites and carry the smallest possible amount of genomic information required to highjack the host cell machinery and perform their replication. This notion, probably inherited from the study of RNA viruses, is being gradually undermined by the discovery of DNA viruses with increasingly large gene content. These viruses also encode a variety of DNA repair functions, presumably slowing down their evolution by preserving their genomes from random alterations. On the other hand, these viruses also encode a majority of proteins without cellular homologs, including many shared only between the closest members of the same family. One may thus question the actual contribution of these anonymous and/or quasi-orphan genes to virus fitness. Genomic comparisons of Marseilleviridae, including a new Marseillevirus isolated in Australia, demonstrate that most of their genes, irrespective of their functions and conservation across families, are evolving under negative selection.

  • Viral Reprogramming of the Daxx Histone H3.3 Chaperone during Early Epstein-Barr Virus Infection [Genome Replication and Regulation of Viral Gene Expression]

  • Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive cochaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using coimmunoprecipitation and size-exclusion chromatography with highly purified components. FRAP (fluorescence recovery after photobleaching) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence combined with fluorescence in situ hybridization that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression.

    IMPORTANCE Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent antiviral repressive chromatin and establish chromatin structure permissive for viral gene expression and the establishment of latent infection.

  • Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy [Vaccines and Antiviral Agents]

  • Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.

    IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.

  • Determinants of Efficient Degradation of APOBEC3 Restriction Factors by HIV-1 Vif [Virus-Cell Interactions]

  • The APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces 48K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in 63K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation.

    IMPORTANCE APOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used differing Vif degradation capacities of two Vif variants and various APOBEC3 enzymes with differential sensitivities to Vif to delineate determinants of the APOBEC3-Vif interaction that are required for inducing efficient degradation. Using a combined biochemical and cellular approach we identified that the strength of the APOBEC3-Vif binding interaction and the APOBEC3-Vif interface are determinants for degradation efficiency. Our results highlight the importance of using Vif variants with different degradation potential when delineating mechanisms of Vif-induced APOBEC3 degradation and identify features important for consideration in the development of HIV-1 therapies that disrupt the APOBEC3-Vif interaction.

  • Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses [Cellular Response to Infection]

  • Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-bbeta; in murine and human cells in the presence of an intact E3L gene. IFN-bbeta; induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-aalpha;, IFN-, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo.

    IMPORTANCE Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.

  • Complex Expression of the UL136 Gene of Human Cytomegalovirus Results in Multiple Protein Isoforms with Unique Roles in Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Human cytomegalovirus (HCMV) is a complex DNA virus with a 230-kb genome encoding 170 and up to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb' region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and, possibly, the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibited distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane-bound isoforms of UL136 differed between when they were expressed exogenously and when they were expressed in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection-specific factors. While UL136, like most ULb' genes, was dispensable for replication in fibroblasts, the soluble 23- and 19-kDa isoforms suppressed virus replication. In CD34+ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23- and 19-kDa isoforms resulted in increased replication and a loss of the latency phenotype, similar to the effects of the UL138 latency determinant encoded within the same genetic locus. Our work suggests a complex interplay between the UL136 isoforms which balances viral replication in multiple cell types and likely contributes to the cell type-dependent phenotypes of the UL133/8 locus and the outcome of HCMV infection.

    IMPORTANCE HCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. The lifelong persistence of the virus results in a high seroprevalence worldwide and may contribute to age-related pathologies, such as atherosclerosis. The mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominantly from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34+ HPCs for latency.

  • Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells [Cellular Response to Infection]

  • The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35ggt;Ad7ggt;Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency.

    IMPORTANCE This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions. Furthermore, activation of the cGAS cascade occurred in a cell-specific manner. Activation of the cGAS/STING response did not impact viral replication, and viral immune evasion strategies did not target the cGAS/STING/TBK1/IRF3 cascade. These studies provide novel insight into the early innate recognition response to adenovirus.

  • The VP40 Protein of Marburg Virus Exhibits Impaired Budding and Increased Sensitivity to Human Tetherin following Mouse Adaptation [Virus-Cell Interactions]

  • The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting Janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus [RAVV]) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by coprecipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells.

    IMPORTANCE Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens that cause severe disease in humans and nonhuman primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse adaptation are poorly understood. Our study demonstrates how genetic changes that accrued during mouse adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor.

  • Tumor Necrosis Factor Alpha and Interleukin-6 Facilitate Corneal Lymphangiogenesis in Response to Herpes Simplex Virus 1 Infection [Pathogenesis and Immunity]

  • Herpes simplex virus 1 (HSV-1) is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously shown that HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of vascular endothelial growth factor A (VEGFA) dependent upon expression of VEGFR2 during acute infection. We hypothesized that other factors may be involved in lymphangiogenesis, with proinflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of tumor necrosis factor alpha (TNF-aalpha;) coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as a vehicle control. Consistent with this observation, anti-TNF-aalpha; antibody (Ab) blocked HSV-1-mediated corneal lymphangiogenesis within the first 5 days postinfection. However, TNF-aalpha;-deficient (TNF-aalpha;nndash;/nndash;) mice displayed a level of corneal vessel growth similar to that shown by wild-type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-aalpha;nndash;/nndash; mice was conducted, and it revealed several factors elevated above those found in HSV-1-infected WT mice, including interleukin-1bbeta; (IL-1bbeta;), platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-aalpha;nndash;/nndash; mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1bbeta; or other proangiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-aalpha; and IL-6 promote and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection.

    IMPORTANCE We have identified at least two proinflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-aalpha; as additional proangiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

  • Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region [Genome Replication and Regulation of Viral Gene Expression]

  • The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an "unstructured" linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function.

    IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in the pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

  • Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro [Virus-Cell Interactions]

  • Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids.

    IMPORTANCE The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called "naked" and membrane-associated cytoplasmic alphaherpesvirus capsids.

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    JVI Accepts: Articles Published Ahead of Print

  • Identification of a role for nucleolin in rabies virus infection [PublishAheadOfPrint]

  • Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localise to nucleoli, and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin, and that nucleolin is important to lyssavirus infection.

  • Influenza A virus uses intercellular connections to spread to neighboring cells [PublishAheadOfPrint]

  • In the extracellular environment cell-free virions seek out naiiuml;ve host cells over long distances and between organisms. This is the primary mechanism of spread for most viruses. Here we provide evidence for an alternative pathway previously undescribed for Orthomyxoviruses where the spread of influenza A virus (IAV) infectious cores to neighboring cells can occur within intercellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection. Connected cells have contiguous membranes and the core infectious viral machinery (RNP and polymerase) were present inside the intercellular connections. A live cell movie of GFP-tagged NS1 of IAV shows viral protein moving from one cell to another through an intercellular connection. Movement of tagged protein was saltatory but overall traveled only in one direction. Infectious virus cores can move from one cell to another without budding and release of cell-free virions, as evidenced by finding that whereas a neuraminidase inhibitor alone did not inhibit the development of IAV microplaques, the presence of a neuraminidase inhibitor together with drugs inhibiting actin dynamics, or the microtubule stabilizer taxol precluded microplaque formation. Similar results were also observed with parainfluenza virus 5 (PIV5), a paramyxovirus, when neutralizing antibody was used to block spread by cell-free virions. Intercellular spread of infectious core particles was unaffected or enhanced in the presence of nocodazole for IAV but inhibited for PIV5. The intercellular connections have a core of filamentous actin, which hints toward transport of virus particles through the use of a myosin motor.

    IMPORTANCE Here we describe a new method by which influenza A virus (IAV) spreads from cell to cell: IAV uses tunneling nanotubes. The formation of these connections requires actin dynamics and is enhanced by viral infection and the absence of microtubules. Connected cells appeared to have contiguous membranes and the core infectious viral machinery (RNP and polymerase) were present inside the intercellular connections. Infectious virus cores can move from one cell to another without budding and release of cell-free virions. Similar results were also observed with parainfluenza virus 5 (PIV5).

  • Hepatitis B core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis by suppression of the phosphorylation of mitogen-activated protein kinase kinase 7 [PublishAheadOfPrint]

  • Hepatitis B, which caused by Hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation is the main driving factor of liver fibrosis and cirrhosis in chronic hepatitis B. Pro-inflammatory tumor necrosis factor (TNF)-aalpha; has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B core (HBc) protein made cells susceptible to TNF-aalpha;-induced apoptosis. We found that receptor of activated protein kinase C (RACK) 1 interacted with HBc by tandem affinity purification and mass spectrometry. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-aalpha;-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase. In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-aalpha;-induced apoptosis, while overexpression of RACK1 counteracted the pro-apoptotic activity of HBc. Capsid particle formation was not obligatory for HBc pro-apoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-aalpha;-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus a first indication of the pathogenic effects of HBc in liver injury during hepatitis B.

    Importance: Our study revealed a previously unappreciated role of HBc in TNF-aalpha;-mediated apoptosis. The pro-apoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, severe hepatitis-associated HBV variants may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-aalpha;-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

  • The Enterovirus 71 procapsid binds neutralizing antibodies and rescues virus infection in vitro [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability of causing severe disease and death, especially in young children. Although several vaccines are currently in clinical trials no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71 infected cells: an expanded empty capsid, sometimes called procapsid, and the infectious virus. Specifically an immunodominant epitope of EV71 that maps to the virus canyon is structurally different between the procapsid and virus. This structure function study shows that the procapsid can sequester antibodies thus enhancing EV71 infection in vitro. The results presented here suggest that due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics.

    IMPORTANCE In a picornavirus infection both an infectious and a non-infectious empty capsid, sometimes referred to as procapsid, are produced. It was novel to discover that the procapsid form of enterovirus 71 (EV71) was expanded and antigenically distinct from the infectious virus. Previously it had been supposed that this empty capsid was an off pathway dead end, or at best served as storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much non-infectious capsid. Here we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

  • Non-Muscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry [PublishAheadOfPrint]

  • Non-muscle myosin heavy chain IIA (NMHC-IIA) has been reported to function as a herpes simplex virus 1 (HSV-1) entry co-receptor by interacting with viral envelope glycoprotein B (gB). Vertebrates have three genetically distinct isoforms of the NMHC-II, designated NMHC-IIA, NMHC-IIB and NMHC-IIC. COS cells, which are readily infected by HSV-1, do not express NMHC-IIA but do express NMHC-IIB. This observation prompted us to investigate whether NMHC-IIB might associate with HSV-1 gB and be involved in an HSV-1 entry like NMHC-IIA. In these studies, we show that: (i) NMHC-IIB co-precipitated with gB in COS-1 cells upon HSV-1 entry; (ii) a specific inhibitor of myosin light chain kinase inhibited both cell-surface expression of NMHC-IIB in COS-1 cells upon HSV-1 entry as well as HSV-1 infection as reported with NMHC-IIA; (iii) overexpression of mouse NMHC-IIB in IC21 cells significantly increased their susceptibility to HSV-1 infection; (iv) knock-down of NMHC-IIB in COS-1 cells inhibited HSV-1 infection as well as cell-cell fusion mediated by HSV-1 envelope glycoproteins. These results supported the hypothesis that, like NMHC-IIA, NMHC-IIB associated with HSV-1 gB and mediated HSV-1 entry.

    Importance Herpes simplex virus 1 (HSV-1) was reported to utilize non-muscle myosin heavy chain IIA (NMHC-IIA) as an entry co-receptor associating with gB. Vertebrates have three genetically distinct isoforms of NMHC-II. In these isoforms, NMHC-IIB is of special interest since it highly expresses in neuronal tissue, one of the most important cellular targets of HSV-1 in vivo. In this study, we demonstrated that the ability to mediate HSV-1 entry appeared to be conserved in NMHC-II isoforms. These results may provide an insight into the mechanism by which HSV-1 infects a wide variety of cell types in vivo.

  • Characterization of a proteolytically stable D-peptide that suppresses Herpes simplex virus-1 infection: Implications for the development of entry-based antiviral therapy [PublishAheadOfPrint]

  • Uncontrolled herpes simplex virus type-1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo.

  • Human Noroviruses' Fondness of Histo-Blood Group Antigens [PublishAheadOfPrint]

  • Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs) and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions to different HBGA types. Here, we show the high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants in 2004, 2006, and 2012, co-crystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A-type, and B-type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391-395) was elegantly repositioned in order to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications.

    Importance: Our data provides a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain HBGA interactions we identified were negative in earlier ELISA-based studies. Altogether, we showed that the GII.4 norovirus P domains could accommodate numerous HBGA types.

  • Interferon-induced Ifit proteins: Their role in viral pathogenesis [PublishAheadOfPrint]

  • A major component of the protective antiviral host defense is contributed by the intracellular actions of the proteins encoded by interferon-stimulated genes (ISG); amongst these are the Interferon-induced proteins with tetratricopeptide repeats (IFIT), consisting of four members in human and three in mouse. IFIT proteins do not have any known enzyme activity; instead, they inhibit virus replication by binding and regulating the functions of cellular and viral proteins and RNAs. Although all IFITs are comprised of multiple copies of the degenerate tetratricopeptide repeats, their distinct tertiary structures enable them to bind different partners and affect host-virus interactions differently. The recent use of Ifit knock-out mouse models has revealed novel antiviral functions of these proteins and new insights into the specificities of ISG actions. This article focuses on human and murine IFIT1 and IFIT2 by reviewing their mechanisms of action, their critical roles in protecting mice from viral pathogenesis and viral strategies to evade IFIT action.

  • Receptor recognition mechanisms of coronaviruses - a decade of structural studies [PublishAheadOfPrint]

  • Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection, and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors, and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses, and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general.

  • The NSP3 Macrodomain Promotes Virulence in Mice With Coronavirus-induced Encephalitis [PublishAheadOfPrint]

  • All coronaviruses encode for a macrodomain containing ADP-ribose-1"-phosphatase (ADRP) activity within the N-terminus of non-structural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reduced lethality, weight loss, viral titers, pro-inflammatory cytokine and chemokine expression, and immune cell infiltration in the brain when compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 post-infection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus, as all N1347A-infected mice survived the infection. However, the adaptive immune system was required for protection, since N1347A virus was able to cause lethal encephalitis in RAG1 (recombination activation gene1)-/- knockout mice, although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV, and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis.

    IMPORTANCE Coronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain, and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1"-phosphatase activity; however, its function during infection remains unclear as does why coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies, and may give further insight into the role of macrodomain proteins in the host cell, since these proteins are found in all living organisms.

  • Vaccine-induced protection against orthopoxvirus infection is mediated through the combined functions of CD4 T cell-dependent antibody and CD8 T cell responses [PublishAheadOfPrint]

  • Antibody production by B cells, in the absence of CD4 T cell help, has been shown to be necessary and sufficient for protection against secondary orthopoxvirus infections. This conclusion is based on short-term depletion of leukocyte subsets in vaccinated animals in addition to passive transfer of immune serum to naiiuml;ve hosts that are subsequently protected from lethal orthopoxvirus infection. Here we show that CD4 T cell help is necessary for neutralizing antibody production and virus control during a secondary ectromelia virus (ECTV) infection. A crucial role for CD4 T cells was revealed when depletion of this subset was extended beyond the acute phase of infection. Sustained depletion of CD4 T cells over several weeks in vaccinated animals during a secondary infection resulted in gradual diminution of B cell responses including neutralizing antibody, contemporaneous with a corresponding increase in viral load. Long-term elimination of CD8 T cells alone delayed virus clearance but prolonged depletion of both CD4 and CD8 T cells resulted in death associated with uncontrolled virus replication. In the absence of CD4 T cells, perforin- and granzyme A and B-dependent effector functions of CD8 T cells became critical. Our data therefore show that both CD4 T cell help for antibody production and CD8 T cell effector function are critical for protection against secondary OPV infection. These results are consistent with the notion that the effectiveness of the smallpox vaccine is related to its capacity to induce both B and T cell memory.

    IMPORTANCE Smallpox eradication through vaccination is one of the most successful public health endeavors of modern medicine. The use of various orthopoxvirus (OPV) models to elucidate correlates of vaccine-induced protective immunity show that antibody is critical for protection against secondary infection whereas the role of T cells is unclear. Short-term leukocyte subset depletion in vaccinated animals or transfer of immune serum to naiiuml;ve, immunocompetent hosts indicates that antibody alone is necessary and sufficient for protection. We show here that long-term depletion of CD4 T cells over several weeks in vaccinated animals during secondary OPV challenge reveals an important role for CD4 T cell-dependent antibody responses in effective virus control. Prolonged elimination of CD8 T cells alone delayed virus clearance but depletion of both T cell subsets resulted in death associated with uncontrolled virus replication. Thus, vaccinated individuals who subsequently acquire T cell deficiencies may not be protected against secondary OPV infection.

  • The level of viral infection of antigen-presenting cells is critical for the development of TMEV-induced demyelinating disease [PublishAheadOfPrint]

  • Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carry H-2s MHC genes instead of H-2b MHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1 and robust Th17 type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response. In vitro studies using APCs from B6.S and SJL mice show that TLR2, 3, 4, and 7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective vs. pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases.

    Importance: This study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes, which are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.

  • Lethal co-infection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung, and reduces germinal center B cells, T follicular helper cells and plasma cells in mediastinal lymph node [PublishAheadOfPrint]

  • Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how the secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact on the humoral immune response to the initial influenza virus infection in a lethal co-infection mouse model. When compared to mice infected with influenza virus alone, mice co-infected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal co-infection significantly increased virus titer, bacterial cell counts and decreased the level of virus specific IgG, IgM and IgA, as well as the number of B cells, CD4 T cells and plasma cells. In spleen, lethal co-infection significantly reduced the size and weight of spleen, as well as the B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal co-infection significantly decreased germinal center B cells, T follicular helper cells and plasma cells. Adoptive transfer of influenza virus-specific immune serum to co-infected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, co-infection reduced the B cell response to influenza virus. This study helps us to understand the modulation of B cell response to influenza virus during the lethal co-infection.

    Importance: Secondary pneumococcal infection after influenza is an important clinical issue that often results in excess mortality. Since antibodies are key mediator of protection, this study aims to examine the antibody response to influenza virus, and demonstrated that lethal co-infection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of B cell response in the context of co-infection.

  • Macropinocytosis is the Entry Mechanism of Amphotropic Murine Leukemia Virus (A-MLV) [PublishAheadOfPrint]

  • The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV does not internalize by caveolae or other pinocytic mechanism, but by macropinocytosis. Thus A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, or NIH-3T3 cells knocked-down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH-3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations only mildly or not at all affected the infection of viruses that use clathrin coated-pit endocytosis or other pathways for entry. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of AMLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that AMLV use macropinocytosis as the predominant entry portal into cells.

    IMPORTANCE Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutical schemes and methods of drug delivery. We show here that Murine Leukemia Virus (A-MLV) pseudotyped with the amphotropic (expands the host range to many mammalian cells) envelope protein gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 mmu;m) following collapse of cell surface protrusions and membrane scission. We use drugs or introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection correlate, and provide both light and electron microscopical evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals including caveolae, previously suggested to internalize A-MLV.

  • Nipah Virus Attachment Glycoprotein Stalk C-terminal Region Links Receptor Binding to Fusion Triggering [PublishAheadOfPrint]

  • Membrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane integral viral proteins, the attachment (HN or H or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F, and the specific events that link receptor binding to F triggering, are unknown. In the present study we identified a NiV-G stalk C-terminal region (aa 159-163) that is important for multiple G functions including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. Based on these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk, and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion.

    Importance: The emergent deadly Nipah and Hendra viruses belong to the Henipavirus genus in the Paramyxoviridae family. NiV infections target endothelial cells and neurons and, in humans, result in 40-75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause syncytia formation. After attaching to the host cell receptor, henipaviruses enter the target cell via direct viral-cell membrane fusion mediated by two membrane glycoproteins: the attachment protein (G) and the fusion protein (F). In this study, we identified and characterized a region in the NiV-G stalk C-terminal domain that links receptor binding to fusion triggering via several important glycoprotein functions. These findings advance our understanding of the membrane fusion triggering mechanism(s) for the henipaviruses and the paramyxoviruses.

  • SAMD9 is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses [PublishAheadOfPrint]

  • We show that SAMD9 is an innate host anti-viral stress response element that participates in the formation of anti-viral granules. Poxviruses, myxoma virus and vaccinia virus specifically, utilize virus-encoded host range factor(s), such as members of the C7L superfamily, to antagonize SAMD9 to prevent the granule formation in an eIF2aalpha;-independent manner. When SAMD9 is stimulated due to failure of the viral antagonism during infection, the resulting anti-viral granules exhibit properties different from canonical stress granules.

  • Unraveling the web of viroinformatics: computational tools and databases in viral research [PublishAheadOfPrint]

  • The beginning of the second century of research in the field of virology (the first virus was discovered in 1898) was marked with its amalgamation with bioinformatics resulting in the birth of a new domain - viroinformatics. The availability of more than 100 web servers and databases embracing all or specific viruses (for example dengue, influenza, hepatitis, HIV, HFV, HPV, West Nile etc.) as well as distinct applications (comparative/diversity analysis, viral recombination, siRNA/shRNA/miRNA studies, RNA folding, protein-protein interaction, structural analysis, phylotyping/genotyping) will definitely aid the development of effective drugs and vaccines. However, the information about their access and utility is not available at any single source/platform. Therefore, a compendium of various computational tools/resources dedicated specifically to virology is presented in this article.

  • HYDROPHOBIC AND CHARGED RESIDUES IN THE C-TERMINAL ARM OF HEPATITIS C VIRUS RNA-DEPENDENT RNA POLYMERASE REGULATE INITIATION AND ELONGATION. [PublishAheadOfPrint]

  • The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a bbeta;-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation and elongation. We constructed a series of bbeta;-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation assays but a marked increase in primer extension activities indicating an ability to form more stable elongation complexes with long primer:template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favours the elongation process but hampers the initiation steps. A UTP co-crystal structure of one mutant shows, in contrast to wild type protein, several alternate conformations of the substrate confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a sub-genomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that correct positioning of both the bbeta;-loop and C-terminal arm play an essential role during initiation and in HCV replication in general.

    IMPORTANCE HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections and necessitates a thorough understanding of the functional roles of the various structural features on the RdRp. Here we show that even highly conservative changes, e.g. Tyr -ggt; Phe or Asp -ggt; Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

  • Abortive intrabronchial infection of rhesus macaques with varicella zoster virus provides partial protection against simian varicella virus challenge. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is a human neurotropic alphaherpesvirus and the etiological agent of varicella (chickenpox) and herpes zoster (HZ, shingles). Previously, inoculation of monkeys via the subcutaneous, intratracheal, intravenous or oral-nasal-conjunctival routes did not recapitulate all the hallmarks of VZV infection including varicella, immunity, latency and reactivation. Intrabronchial inoculation of rhesus macaques (RMs) with simian varicella virus (SVV), a homolog of VZV, recapitulates virologic and immunologic hallmarks of VZV infection in humans. Given that VZV is acquired primarily via the respiratory route, we investigated whether intrabronchial inoculation of RMs with VZV would result in a robust model. Despite the lack of varicella and viral replication in either the lungs or whole blood, all four RMs generated an immune response characterized by generation of VZV-specific antibodies and T cells. Two of 4 VZV inoculated RMs were challenged with SVV to determine cross-protection. VZV-immune RMs displayed no varicella rash, decreased SVV viral loads as well as earlier and stronger humoral and cellular immune responses compared to controls. In contrast to SVV, no VZV DNA was detected in sensory ganglia at necropsy. In summary, following an abortive VZV infection, RMs developed an adaptive immune response that confers partial protection against SVV challenge. These data suggest that a replication incompetent VZV vaccine that does not establish latency may provide sufficient protection against VZV disease and that VZV vaccination of RMs followed by SVV challenge provides a model to evaluate new vaccines and therapeutics against VZV.

    Importance Although VZV vaccine strain Oka is attenuated, it can cause mild varicella, establish latency, and in rare cases reactivate to cause herpes zoster (HZ). Moreover, studies suggest that the HZ vaccine (Zostavax) only confers short-lived immunity. Development of more efficacious vaccines would be facilitated by a robust animal model of VZV infection. Data presented in this manuscript show that intrabronchial inoculation of rhesus macaques (RMs) with VZV resulted in an abortive VZV infection. Nevertheless, all animals generated a humoral and cellular immune response that conferred partial cross-protection against simian varicella virus (SVV) challenge. Additionally, VZV DNA was not detected in the sensory ganglia suggesting that viremia might be required for the establishment of latency. Therefore, VZV vaccination of RMs followed by SVV challenge is a model that will support the development of vaccines that boost protective T cell responses against VZV.

  • Early activation of teleost B cells in response to rhabdovirus infection [PublishAheadOfPrint]

  • To date, the response of B cells to specific pathogens has been only scarcely addressed in teleost. In the current work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM+ cells although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM+ cell functionality comparing these effects to those elicited by a TLR3 ligand, Poly I:C. We found that Poly I:C and VHSV significantly up-regulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM+ cells. Further effects included the up-regulated transcription of the CK5B chemokine. Significant inhibition of some of these effects in presence of bafilomicyn A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and initiation of viral transcription. VHSV also provoked the activation of NF-B and the up-regulation of MHC-II cell surface expression on IgM+ cells, that along with an increased transcription of the co-stimulatory molecules CD80/86 and CD83 pointed to a VHSV-induced IgM+ cell activation towards an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM+ cell proliferation, a consistent effect on IgM+ cell survival was detected.

    IMPORTANCE Innate immune responses to pathogens established through their recognition by PRRs have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleost, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects in B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-B and increased IgM+ cell survival. Interestingly, VHSV activated B lymphocytes towards an antigen-presenting profile, suggesting an important role of IgM+ cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

  • Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors [PublishAheadOfPrint]

  • Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses are overlapping, and both exhibit pre-existing humoral and cellular immunity in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. These novel rhesus adenovirus vectors exhibited extremely low seroprevalence in sub-Saharan human populations and proved comparably immunogenic to human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

    Importance Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this manuscript, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

  • The HIV-1 nucleocapsid protein recruits negatively charged lipids on binding to lipid membranes [PublishAheadOfPrint]

  • The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC) and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand, and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (~ 107 M-1) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol- (4, 5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM.

    IMPORTANCE The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or a domain of the Gag precursor also interacts with host proteins, only few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and evidenced that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

  • Proteomic analysis of ubiquitin-like post-translational modifications induced by the adenovirus E4-ORF3 protein [PublishAheadOfPrint]

  • Viruses interact with and regulate many host metabolic pathways in order to advance the viral life cycle and counteract intrinsic and extrinsic antiviral responses. The human Adenovirus (Ad) early protein, E4-ORF3, forms a unique scaffold throughout the nuclei of infected cells and inhibits multiple antiviral defenses including a DNA damage response (DDR) and an interferon response. We previously reported that the Ad5 E4-ORF3 protein induces sumoylation of Mre11 and Nbs1, which are essential for the DDR, and their relocalization into E4-ORF3-induced nuclear inclusions is required for this modification to occur. In this study, we sought to analyze a global change in ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, by the Ad5 E4-ORF3 protein and identify new substrates with these modifications. By a comparative proteome-wide approach utilizing immunoprecipitation/mass spectrometry, we found that Ubl modifications of 166 statistically significant lysine sites in 51 proteins are affected by E4-ORF3 and the proteome of modifications spans a diverse range of cellular functions. Ubl modifications of 92% of these identified sites were increased by E4-ORF3. We further analyzed SUMO3 conjugation of several identified proteins. Our findings demonstrate a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and reveal new SUMO3 substrates induced by E4-ORF3.

    IMPORTANCE The Adenovirus E4-ORF3 protein induces dynamic structural changes in the nuclei of infected cells and counteracts host antiviral responses. One of the mechanisms that accounts for this process is the relocalization and sequestration of cellular proteins into an E4-ORF3 nuclear scaffold, but little is known how this small viral protein affects diverse cellular responses. In this study, we analyzed for the first time the global pattern of Ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, altered by E4-ORF3 expression. The results suggest a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and reveal new SUMO3 substrates targeted by E4-ORF3. Our findings propose Ubl modifications as a new mechanism by which E4-ORF3 may modulate cellular protein functions in addition to subnuclear relocalization.

  • The Large Tegument Protein pUL36 is Essential for Formation of the Capsid Vertex Specific Component at the Capsid-Tegument Interface of HSV-1. [PublishAheadOfPrint]

  • Herpesviruses have a characteristic particle structure comprising an icosahedral capsid, which contains the DNA genome and is in turn surrounded by a proteinaceous tegument layer and a lipid envelope. In herpes simplex virus the interaction between the capsid and tegument is limited to the capsid vertices and involves two minor capsid proteins, pUL17 and pUL25, and the large, inner tegument protein, pUL36. pUL17 and pUL25 form a heterodimeric structure, the capsid vertex specific component (CVSC), that lies on top of the peripentonal triplexes, while pUL36 has been reported to connect the CVSC to the penton. In this study we use virus mutants with deletions in the genes for pUL36 and another inner tegument protein, pUL37, to analyse the contributions of these proteins to CVSC structure. Using electron cryo-microscopy and icosahedral reconstruction of mutants that express pUL17 and pUL25, but not pUL36, we show that, in contrast to accepted models, the CVSC is not formed from pUL17 and pUL25 on their own but requires a contribution from pUL36. In addition, the presence of full-length pUL36 results in weak density that extends the CVSC towards the penton, suggesting, either that this extra density is formed directly by pUL36, or that pUL36 is stabilising other components of the vertex-tegument interface.

    IMPORTANCE Herpesviruses have complex particles that are formed as a result of a carefully controlled sequence of assembly steps. The nature of the interaction between two of the major particle compartments, the icosahedral capsid and the amorphous tegument, has been extensively studied, but the identity of the interacting proteins and their roles in forming the connections are still unclear. In this study we use electron microscopy and 3D reconstruction to analyse virus particles formed by mutants that do not express particular interacting proteins. We show that the largest viral protein, pUL36, which occupies the layer of tegument closest to the capsid, is essential for formation of structurally normal connections to the capsid. This demonstrates the importance of pUL36 in the initial stages of tegument addition and provides new insights into the process of virus particle assembly.

  • BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex to Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins [PublishAheadOfPrint]

  • BGLF4 kinase, the only Ser/Thr protein kinase encoded by EBV, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153, independent of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin-bbeta; was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found BGLF4 promoted the nuclear import of several non-NLS containing EBV proteins, including the viral DNA replicating enzymes BSLF1, BBLF2/3 and BBLF4 and the major capsid protein (VCA), in co-transfected cells. Data here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import promoting activity was found in cells expressing the BGLF4 homologs of another two gamma-herpesviruses, but not those from alpha- and beta-herpesviruses.

    Importance During lytic replication, many EBV encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical-NLS signals are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.

  • GBF1- and ACBD3-independent recruitment of PI4KIII{beta} to replication sites by rhinovirus 3A proteins [PublishAheadOfPrint]

  • PI4KIIIbbeta; recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses like poliovirus and coxsackievirus recruit PI4KIIIbbeta; to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIbbeta; to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although previously HRV 3A was shown to interact with ACBD3, our data suggest that PI4KIIIbbeta; recruitment occurred independently of both GBF1 and ACBD3.

  • The evolution and evolutionary relationships between extant vaccinia virus strains [PublishAheadOfPrint]

  • Although vaccinia virus (VACV) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of VACV are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. Here, we sequenced additional VACV strains that represent either relatively pristine examples of old vaccines (e.g. Dryvax, Lister, and Tashkent) or which have been subjected to additional laboratory passage (e.g. IHD-W and WR). These genome sequences were compared with those previously reported for other VACV as well as other orthopoxviruses. These extant VACV do not always cluster in simple phylogenetic trees that are aligned with the known historical relationships between these strains. Rather, the pattern of deletions suggests that all existing strains likely come from a complex stock of viruses that has been passaged, distributed, and randomly sampled over time, thus obscuring simple historical or geographic links. We examined surviving non-clonal vaccine stocks, like Dryvax, which continue to harbor larger and now rare variants including one we have designated "clone DPP25". DPP25 encodes genes not found in most VACV strains including an ankyrin-F-box protein, a homolog of the variola virus (Bangladesh) B18R gene, which we show can be deleted without affecting virulence in mice. We propose a simple common mechanism by which recombination of a larger and hypothetical DPP25-like ancestral strain, combined with selection for retention of critically important genes near the terminal inverted repeat boundaries (vaccinia growth factor gene and an interferon aalpha;/bbeta; receptor homolog), could produce all known VACV variants.

    IMPORTANCE Smallpox was eradicated using a combination of intensive disease surveillance and vaccination using vaccinia virus (VACV). Interestingly, little is known about the historical relationships between different strains of vaccinia virus (VACV) and how these viruses may have evolved from a common ancestral strain. To understand these relationships additional strains were sequenced and compared to existing strains of VACV as well as other Orthopoxviruses using whole genome sequence alignments. Extant strains of VACV didn't always cluster in simple phylogenetic trees based on known historical relationships between these strains. Based on these findings it is possible that all existing strains of VACV could derive from a single complex stock of viruses that has been passaged, distributed, and sampled over time.

  • Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters [PublishAheadOfPrint]

  • EBV maintains a life-long latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors such as BLIMP1 that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV(+) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic-gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nt -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited TPA-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately-early genes, BZLF1 and BRLF1.

    Importance: This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpes virus, Epstein-Barr virus (EBV), out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate-early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

  • Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge [PublishAheadOfPrint]

  • Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still missing. Here, the properties of novel recombinant SBV-mutants lacking the non-structural proteins NSs (rSBVNSs), NSm (rSBVNSm) or both of them (rSBVNSs/NSm) were tested in vitro and in vivo in type I interferon receptor knock-out mice (IFNAR-/-) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both non-structural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells rSBVNSs and rSBVNSm replicated to comparable level as the parental rSBV, the double mutant virus, however, showed a mild growth defect resulting in lower final virus titers. Additionally, both mutants with a NSs-deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR-/- mice, all mutants were virulent, with the highest mortality rate for rSBVNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viraemia and seroconversion comparable to wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both non-structural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV-vaccines which could be also a model for other viruses of the Simbu-serogroup and related orthobunyaviruses.

    Importance SBV induces only mild clinical signs in adult ruminants, but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated, that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus and, in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine represents also a first model for a broad range of related viruses, and is of high importance to the field.

  • p38MAPK and MK2 pathways are important for the differentiation-dependent HPV life cycle [PublishAheadOfPrint]

  • Amplification of human papillomaviruses (HPV) is dependent on the ATM DNA damage pathway. In cells with impaired p53 activity, DNA damage repair requires the activation of p38MAPK along with MAPKAP kinase 2 (MK2). In HPV positive cells, phosphorylation of p38 and MK2 proteins was induced along with relocalization to the cytoplasm. Treatment with MK2 or p38 inhibitors blocked HPV genome amplification identifying the p38/MK2 pathway as a key regulator of the HPV life cycle.

  • Adeno-associated virus serotypes 1 and 5 - antibody complex structures reveal evolutionary commonalities in parvovirus antigenic reactivity [PublishAheadOfPrint]

  • The clinical utility of the Adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from pre-existing antibodies is detrimental to AAV transduction efficiency. Hence, mapping AAV antigenic sites and engineering neutralization-escaping vectors is important for improving clinical efficacy. We report the structures for four AAV:monoclonal antibody fragment complexes: AAV1:ADK1a, AAV1:ADK1b, AAV5:ADK5a, and AAV5:ADK5b, determined by cryo-electron microscopy and image reconstruction to ~11-12 AAring; resolution. Pseudo-atomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis, the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap with the two dominant epitopes reported for autonomous parvoviruses. Thus rather than amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions.

    Importance The Adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has pre-existing AAV capsid antibodies and therefore must be excluded from clinical trials or re-administration of vector. This manuscript represents our continuing efforts to understand the antigenic structure of the AAVs, specifically to obtain a picture of the "polyclonal" reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryo-electron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide essential information for generating antigenic escape vectors towards overcoming a critical challenge remaining in optimization of this highly promising vector delivery system.

  • Identification of the physiological gene targets of the essential lytic replicative KSHV ORF57 protein. [PublishAheadOfPrint]

  • The KSHV ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57 null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, qPCR, Northern blotting and RACE methods. Comparison of gene expression from a ORF57 KSHV recombinant, rescued ORF57 KSHV recombinant and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57 dependent genes, ORF57 regulation was promoter and polyadenylation signal-independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified as critically requiring ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy.

  • Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214 [PublishAheadOfPrint]

  • In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNAi to deplete cells of either Nup214 or Nup358, the two major FG repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of pVII, indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137 amino acid segment in N-terminal domain of Nup214. Together, our results have identified a specific region within the N-terminus of Nup214 that acts as a direct NPC binding site for AdV.

    Importance AdV, which have the largest genome of non-enveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137 amino acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer, and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

  • Global displacement of canine parvovirus by a host-adapted variant: A structural comparison between pandemic viruses with distinct host ranges [PublishAheadOfPrint]

  • Canine parvovirus (CPV-2) emerged in 1978 and spread worldwide within two years. Subsequently, CPV-2 was completely replaced by the variant CPV-2a, which is characterized by four specific capsid (VP2) mutations. The X-ray crystal structure of the CPV-2a capsid shows that each mutation confers small local changes. The loss of a hydrogen bond and introduction of a glycine residue likely introduce flexibility to sites that control interactions with the host receptor, antibodies, and sialic acids.

  • The Focal Adhesion kinase is involved in rabies virus infection through its interaction with the viral phosphoprotein P [PublishAheadOfPrint]

  • The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition RABV P has been identified as an interferon antagonist. Here, a yeast two hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the domain 106-131 corresponding to the dimerization domain of P and the C-ter domain of FAK containing the proline rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by co-immunoprecipitation and co-localization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Arg to Ala in position 109 (PR109A) in P, which did not interact with FAK, is affected at a post-transcriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RAV with FAK which positively regulates infection.

    Importance Rabies virus exhibits small-sized genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional such as the phosphoprotein P. Indeed, we and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the Focal Adhesion Kinase (FAK). Our data shed light on the implication of FAK in RAV infection and provide evidence that P-FAK interaction has a proviral function.

  • Syntaxin 5-Dependent Retrograde Transport to the Golgi is Required for AAV Transduction. [PublishAheadOfPrint]

  • Intracellular transport of recombinant Adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasmmmdash;allowing subsequent nuclear import and the initiation of gene expressionmmdash;remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus, and that Golgi disruption caused by the drug brefeldin A efficiently blocks AAV2 transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of Golgi transport for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a non-classical retrograde transport pathway that is independent of retromer, late endosomes and recycling endosomes. AAV2 transduction is unaffected by knock-down of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by siRNA silencing or treatment with Retro-2cycl strongly decrease AAV2 transduction and Golgi transport. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.

    IMPORTANCE Gene therapy constitutes a promising approach for treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne Muscular Dystrophy, hemophilia, heart failure, Parkinson disease and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from known Golgi-targeted cargos, and we raise the possibility that this non-classical pathway is shared by most AAV variants, regardless of their attachment receptors.

  • Identification and mutagenesis of the AAV5 sialic acid binding region [PublishAheadOfPrint]

  • As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of AAVs utilize sialic acid (SIA) for binding and transduction, this virus:carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the threefold axis of symmetry, while the second site was located under the HI loop close to the fivefold axis. Mutagenesis of amino acids 569 and 585 or 587 within the threefold depression resulted in elimination or alteration in SIA dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the threefold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polycloncal antibody raised against the wild type virus.

    Importance Despite that a majority of AAVs utilize sialic acid for binding and transduction, this virus: carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and study using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild type virus.

  • A live attenuated equine H3N8 influenza vaccine is highly immunogenic and efficacious in mice and ferrets [PublishAheadOfPrint]

  • Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infection of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans.

    IMPORTANCE Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts and spreads in humans causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and protected mice and ferrets against homologous and heterologous EIV.

  • Andes Virus Nucleocapsid Protein Interrupts PKR Dimerization to Counteract The Host Interference in Viral Protein Synthesis. [PublishAheadOfPrint]

  • Pathogenic hantaviruses delay type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon stimulated genes, observed during later stages of hantavirus infection fails to combat the virus replication in infected cells. Protein kinase R, a classical interferon stimulated gene product phosphorylates the eukaryotic translation initiation factor eIF2aalpha; and causes translation shutdown to create roadblocks for the synthesis of viral proteins. The PKR induced translation shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus infected cells do not undergo translation shutdown and fail to establish an antiviral state during the course of viral infection. Here we show for the first time that Andes virus infection induced PKR over-expression. However, the over-expressed PKR was not active due to significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here have established hantavirus nucleocapsid protein as a new PKR inhibitor. These studies have provided mechanistic insights for hantavirus resistance to host interferon response and have solved the puzzle for the lack of translation shutdown observed in hantavirus infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade PKR antiviral response for survival. We envision that evasion of PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and survive in infected hosts having multifaceted antiviral defense.

    Importance Protein kinase R, a versatile antiviral host factor shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported in this manuscript reveal that hantavirus nucleocapsid protein counteracts PKR antiviral response by inhibiting PKR dimerization, required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus infected cells prevents the PKR induced host translation shutdown to ensure the continuous synthesis of viral proteins, required for efficient virus replication.

  • Identification of RISC-associated adenoviral miRNAs, a subset of their direct targets, and global changes in the targetome upon lytic Ad5 infection [PublishAheadOfPrint]

  • Adenoviruses encode a set of highly abundant miRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger non-coding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant MOIs. In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the post-transcriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells we determined changes in cellular miRNA levels in both total RNA/RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide, trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the 2 most abundant isoforms in RISCs of lytically infected A549 cells.

    Importance Viral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC-targetome during lytic adenovirus infection, and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net-detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

  • Microparticles in Dengue Infection Provide a Novel Biomarker to Predict Severe Clinical Outcomes [PublishAheadOfPrint]

  • Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis whereas increased MP generation is linked to many pathologic conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens of patients with different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement opsonized NS1-anti NS1 immune complexes bound to erythrocytes via complement receptor 1 triggered MP shedding in vitro nndash; a process that could explain increased RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever.

    Importance Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs) - tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patient blood are changed in response to DENV infection. Infection of cells led to programed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with bleeding tendency while increased red blood cell-derived MPs (RMPs) correlated with disease severity. Importantly, the level of RMPs at the early acute period could serve as a biomarker to identify potentially severe patients who require immediate care.

  • Cancer-Causing Human Papillomavirus E6 Proteins Display Major Differences in the Phospho-Regulation of their PDZ Interactions. [PublishAheadOfPrint]

  • Previous studies have shown that that the cancer-causing high risk HPV E6 oncoproteins have PDZ binding potential, an activity which is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions are not constitutive, and can be negatively regulated by phosphorylation within the E6 PDZ binding motif (PBM). In this study we have investigated the differential regulation of the HPV E6 PBM from diverse high-risk HPV types. We show that, depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with PKA or AKT, which in turn inhibits PDZ recognition. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18 and HPV-58, whilst being somewhat weaker or absent from other types such as HPV-31, 33 and 51. In the case of HPV31, PKA phosphorylation occurs within the core of the E6 protein and has no effect on PDZ interactions, and this demonstrates a surprising degree of heterogeneity amongst the different high risk HPV E6 oncoproteins in how they are regulated by different cellular signaling pathways.

    Importance This study demonstrates that the cancer-causing HPV E6 oncoproteins are all subject to post translational modification of their extreme C terminal PDZ binding motif through phosphorylation. However the identity of the kinase is not the same for all HPV types. This demonstrates a very important divergence between these HPVs, and it suggests that changes in cell signalling pathways will have different consequences for different high risk virus infections and their associated malignancies.

  • Dual Pro-inflammatory and Antiviral Properties of Pulmonary Eosinophils in Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Disease [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the role of pulmonary eosinophilia in the antiviral response and in disease pathogenesis is inadequately understood. In vivo studies in mice having eotaxin and/or interleukin (IL)-5 deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease where following challenge there was reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses and altered chemokines (TARC, CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin and/or IL-5 deficient mice. Adoptive transfer of eosinophils to FI-RSV immunized eotaxin and IL-5 deficient (double deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in viral load.

    Importance This study is critical to understanding the mechanisms attributable to RSV vaccine enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine enhanced disease. The findings suggest that, while eosinophils within a Th2-polarized inflammatory response may have pathologic features, they are also effective mediators of antiviral defence.

  • Fruits of Virus Discovery: New Pathogens and New Experimental Models [PublishAheadOfPrint]

  • The application of high-throughput sequencing has lead to a tremendous increase in the rate of discovery of viral sequences. In some instances, novel pathogens have been identified. What has been less well appreciated is that novel virus discoveries in distinct hosts have led to the establishment of unique experimental systems to define host-virus interactions. These new systems have opened new frontiers in the study of fundamental virology and infectious disease.

  • Three novel virophage genomes discovered from Yellowstone Lake metagenomes [PublishAheadOfPrint]

  • Virophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study the genomes of three novel virophages YSLV5, YSLV6 and YSLV7 were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLVs 1-4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767 bp genome with 32 predicted ORFs, YSLV6 has a 24,837 bp genome with 29 predicted ORFs, and YSLV7 has a 23,193 bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLVs 1-4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem.

    Importance This study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5-7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic zone and non-hydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic zone samples, and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages, and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.

  • Electron cryo-tomography studies of maturing HIV-1 particles reveal the assembly pathway of the viral core [PublishAheadOfPrint]

  • To better characterize the assembly of the HIV-1 core, we have used electron cryo-tomography (ECT) to image infected cells and the viral particles cryo-preserved next to them. We observed progressive stages of virus assembly and egress including flower-like flat gag lattice assemblies, hemispherical budding profiles and virus buds linked to the plasma membrane via a thin membrane neck. The population of budded viral particles contains immature, maturation intermediate and mature core morphologies. Structural characteristics of the maturation intermediates suggest that the core assembly pathway involves the formation of a CA sheet that associates with the condensed ribonucleoprotein (RNP) complex. Our analysis also reveals a correlation between RNP localization within the viral particle and the formation of conical cores, suggesting that the RNP helps drive conical core assembly. Our findings support an assembly pathway for the HIV-1 core that begins with a small CA sheet that associates with the RNP to form the core base, followed by polymerization of the CA sheet along one side of the conical core towards the tip and then closure around the body of the cone.

    IMPORTANCE During HIV-1 assembly and release the Gag polyprotein is organized into a signature hexagonal lattice, termed the immature lattice. To become infectious, the newly budded virus must disassemble the immature lattice by proteolyzing Gag, and then reassemble the key proteolytic product, the structural protein p24 (CA), into a distinct, mature hexagonal lattice during a process termed maturation. The mature HIV-1 virus contains a conical capsid that encloses the condensed viral genome at its wide base. Mutations or small molecules that interfere with viral maturation also disrupt viral infectivity. Little is known about the assembly pathway that results in the conical core and genome encapsidation. Here we have used electron cryo-tomography to structurally characterize HIV-1 particles that are actively maturing. Based on the morphologies of core assembly intermediates, we propose that CA forms a sheet-like structure that associates with the condensed viral genome to produce the mature infectious conical core.

  • Abrogation of the interferon response promotes more efficient human cytomegalovirus replication [PublishAheadOfPrint]

  • The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to either block the production of or the response to type I IFNs. In IFN-deficient cells HCMV produced larger plaques, spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.

  • Biological and protective properties of immune sera directed to influenza virus neuraminidase [PublishAheadOfPrint]

  • The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain which is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, this antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in a NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activity of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spreading of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA-recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high quality NA antibodies for controlling influenza virus replication and place emphasis on NA as vaccine antigen.

    Importance The neuraminidase of influenza A viruses is a sialidase which acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera not only inhibited sialidase activity but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed a broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtypic activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next generation influenza virus vaccines.

  • A CryoEM study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment [PublishAheadOfPrint]

  • Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16 we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-EM structures of HPV16 mature and an altered capsid particles were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudo-atomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered capsid-Fab complex map showed that binding of the Fab induced significant conformational changes than were seen in the altered capsid structure alone. These changes included more ordered surface loops, consolidated invading arm structures, and tighter inter-capsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryoEM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyper-stabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites.

    IMPORTANCE Our analysis of the cryoEM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer resulting in enhanced inter-capsomeric interactions evidenced by the more ordered capsid floor and llsquo;invading armrrsquo; structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

  • Two distinct sets of NS2A molecules are responsible for dengue virus RNA synthesis and virion assembly [PublishAheadOfPrint]

  • Flavivirus nonstructural protein 2A (NS2A) plays important roles in both viral RNA synthesis and virion assembly. The molecular details of how NS2A protein modulates the two distinct events have not been defined. To address this question, we have performed a systematic mutagenesis of NS2A using dengue virus serotype 2 (DENV-2) as a model. We identified two sets of NS2A mutations with distinct defects during a viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis. Mechanistically, mutation D125A abolished viral RNA synthesis through blocking the N-terminal cleavage of NS2A protein, leading to an unprocessed NS1-NS2A protein; this result suggests that amino acid D125 (far downstream of the N-terminus of NS2A) may contribute to the recognition of host protease at the NS1-NS2A junction. The other set of NS2A mutations (G11A, E20A, E100A, Q187A, and K188A) specifically impaired virion assembly without significantly affecting viral RNA synthesis. Remarkably, mutants defective in virion assembly could be rescued by trans supplying wild-type NS2A molecules expressed from a replicative replicon, by wild-type NS2A protein expressed alone, by a mutant NS2A (G200A) that is lethal for viral RNA synthesis, or by a different mutant NS2A that is defective in virion assembly. In contrast, none of the mutants defective in viral RNA synthesis could be rescued by trans complementation. Collectively, the results indicate that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis and virion assembly.

    SIGNIFICANCE Dengue virus (DENV) represents the most prevalent mosquito-borne human pathogen. Understanding the replication of DENV is essential for development of vaccine and therapeutics. Here we characterized the function of DENV-2 NS2A using a systematic mutagenesis approach. The mutagenesis results revealed two distinct sets of NS2A mutations: One set of mutations that are defective in viral RNA synthesis; another set of mutations that are defective in virion assembly. Trans complementation analysis showed that mutants defective in viral RNA synthesis could not be rescued by wild-type NS2A; in contrast, mutants defective in virion assembly could be successfully rescued by wild-type NS2A or even by a mutant NS2A that is incompetent to support viral RNA synthesis. These results support a model that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis (located in the viral replication complex) and virion assembly (located in the virion assembly/budding site). The study has confirmed and extended our understanding of the two critical roles of flavivirus NS2A in viral RNA synthesis and virion assembly.

  • Ebola virus transmission in guinea pigs [PublishAheadOfPrint]

  • Ebola virus (EBOV) transmission is currently poorly characterized and thought to occur primarily by direct contact with infectious material; however transmission from swine to nonhuman primates via the respiratory tract has been documented. To establish an EBOV transmission model for performing studies with statistical significance, groups of six guinea pigs (gps) were challenged intranasally (IN) or intraperitoneally (IP) with 10,000 x LD50 of gp-adapted EBOV, and naiiuml;ve gps were then introduced as cage-mates for contact exposure at 1 day post-infection (dpi). Animals were monitored for survival and clinical signs of disease, and quantitated for virus shedding post-exposure. Changes in contact duration of naiiuml;ve gps with infected animals were evaluated for impact on transmission efficiency. Transmission was more efficient from IN compared to IP-challenged gps, with 17% versus 83% of naiiuml;ve gps surviving exposure, respectively. Virus shedding was detected beginning at 3 dpi from both IN- and IP-challenged animals. Contact duration positively correlated with transmission efficiency, and the abrogation of direct contact between infected and naiiuml;ve animals through the erection of a steel mesh is effective at stopping virus spread, provided that infectious animal bedding was absent in the cages. Histopathological and immunohistochemical findings show that IN-infected gps display enhanced lung pathology and EBOV antigen in the trachea, which support increased virus transmission from these animals. The results suggest that IN-challenged gps are more infectious to naiiuml;ve animals than their systemically-infected counterparts, and that transmission occurs through direct contact with infectious materials, including those transported through air movement over short distances.

    Importance Ebola is generally thought to be spread between humans though infectious bodily fluids. However, a study has shown that Ebola can be spread from pigs to monkeys without direct contact. Further studies have been hampered, because an economical animal model for Ebola transmission is not available. To address this, we established a transmission model in guinea pigs, and determined the mechanisms behind virus spread. The survival data, in addition to microscopic examination of lung and trachea sections, show that mucosal infection of guinea pigs is an efficient model for Ebola transmission. Virus spread is increased with longer contact times to an infected animal and is possible without direct contact between an infected and naiiuml;ve host, but can be stopped if infectious materials were absent. These results warrant consideration for the development of future strategies against Ebola transmission, and a better understanding of the parameters involved with virus spread.

  • Specificity of coxsackievirus B3 interaction with human, but not murine, decay accelerating factor (DAF): replacement of a single residue within short consensus repeat 2 prevents virus attachment [PublishAheadOfPrint]

  • Many coxsackievirus B (CVB) isolates bind to human decay accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). However, virus does not interact with murine DAF. To understand why CVB3 binds specifically to human DAF we constructed a series of chimeric molecules in which specific regions of the human DAF molecule were replaced by the corresponding murine sequences. We found that replacement of human with murine SCR2 ablated virus binding to human DAF, as did deletion of human SCR2. Although replacement of human SCR4 had a partial inhibitory effect, deletion of SCR4 had no effect. Within human SCR2, replacement of serine 104 (S104) with the proline residue found in murine DAF eliminated virus binding. Based on the structure of the CVB3-DAF complex determined by cryo-electron microscopy, DAF S104 is in close contact with a viral capsid residue, a threonine at VP1 position 271. Replacement of this capsid residue with larger amino acids specifically eliminated virus attachment to human DAF, but had no effect on attachment to CAR or replication in HeLa cells. Taken together, these results support the current model of virus-DAF interaction, and point to a specific role for VP1-T271 and DAF S104 at the virus-DAF interface.

    IMPORTANCE These results point to a specific role for VP1-T271 and DAF S104 at the interface between CVB3 and DAF-DAF, and demonstrate how subtle structural changes can dramatically influence virus-receptor interactions. In addition, the results support a recent pseudoatomic model of CVB3-DAF interaction obtained by cryo-electron microscopy.

  • Structure-based functional analyses of domains II and III of pseudorabies virus glycoprotein H [PublishAheadOfPrint]

  • Enveloped viruses utilize membrane fusion for entry into, and release from host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB, and a heterodimer of gH and gL. The crystal structures of three gH homologues including pseudorabies virus (PrV) gH revealed four conserved domains. Domain II contains a planar bbeta;-sheet (fence), and a syntaxin-like bundle of three aalpha;-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues, or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent "fence" region or to domain III. Disruption of either of the three aalpha;-helices of the SLB (V250P, V275P, A298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants were also observed after disulfide-linkage of two aalpha;-helices within the SLB (A284C-S291C), or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion.

    Importance: Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the "flap" region in domain IV of PrV gH is important for its function. We now demonstrate that mutations within domain II which interfere with integrity or flexibility of a syntaxin-like three-helix bundle also significantly impair gH function during fusion. These studies provide important insights into the structural requirements of gH for function in fusion.

  • Measles Virus Glycoprotein Complexes Preassemble Intracellularly and Relax During Transport to the Cell Surface in Preparation for Fusion [PublishAheadOfPrint]

  • Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H-stem mutants of progressively shorter length. Conformationally restricted H-stems remained capable of intracellular assembly with standard F and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase a MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in pre-receptor bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic for the morbillivirus genus.

  • The ORF012 gene of Marek's disease virus (MDV) produces a spliced transcript and encodes a novel nuclear phosphoprotein essential for virus growth [PublishAheadOfPrint]

  • Marek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV encodes a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potential and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique-long region. Using reverse transcription PCR we showed that ORF011 and 012 are not individual genes, but encode a single gene through mRNA splicing of a small intron resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defect in vitro and could not be passaged in cultured cells suggesting an essential role for the ORF012 product for virus replication. Further studies revealed that p012 localized to the nucleus in transfected and infected cells and we identified by site-directed mutagenesis and GFP reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23 amino acid sequence at the protein's C-terminus. Nuclear export was blocked using leptomycin B suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution.

    Importance Marek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion US dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle that eventually could help to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C-terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important.

  • Tulane virus recognizes the A-type 3 and B histo-blood group antigens [PublishAheadOfPrint]

  • Tulane virus (TV), the prototype of the Recovirus genus in the calicivirus family, was isolated in the stools of rhesus monkeys and can be cultivated in vitro in monkey kidney cells. TV is genetically closely related to the Norovirus genus and recognizes the histo-blood group antigens (HBGAs) like human noroviruses (NoVs), making it a valuable surrogate for human NoVs. However, the precise structures of HBGAs recognized by TV remain elusive. In this study, we performed binding and blocking experiments on TV with extended HBGA types and showed that, while TV binds all four types (types 1 to 4) of the B antigens, it recognizes only the A-type 3 antigen among four types of A antigens tested. The requirements of HBGAs in TV replication were demonstrated by blocking of TV replication in cell culture using the A-type 3/4 and B saliva samples. Similar results were also observed in oligosaccharide-based blocking assays. Importantly, the previously reported, unexplained increase of TV replication by oligosaccharide in cell-based blocking assays has been clarified, which will facilitate the application of TV as a surrogate for human NoVs.

    Importance. Our understanding on the role of HBGAs in NoV infection has been significantly advanced in the past decade but direct evidence on HBGAs as a receptor for NoVs remains lacking due to a lack of a NoV cell culture. TV recognizes HBGAs and can replicate in vitro, providing a valuable surrogate for human NoVs. However, TV binds to some but not all saliva samples from the A positive individuals and an unexplained result on synthetic oligosaccharides in blocking TV binding have been reported. These issues now have been resolved in this study.

  • Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra virus and enables real-time monitoring of viral spread in small animal models of henipavirus infection. [PublishAheadOfPrint]

  • Nipah (NiV) and Hendra (HeV) viruses are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by gge;3 logs, which offset the difficulty of generating multiple recombinants under constraining BSL-4 conditions. We then substituted singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the early higher replicative titers of HeV-M-bearing chimeras, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a Firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon-aalpha;/bbeta; receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and respiratory tract replication, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipa-like viruses.

    Importance Nipah (NiV) and Hendra (HeV) viruses are recently emergent, zoonotic, and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated generation of reporter-expressing viruses and recombinant NiV-HeV chimeras substituted in the genes responsible for viral exit (M, critical for assembly and budding) and viral entry (attachment (G) and fusion (F)). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice, the first time such in vivo imaging has been performed under BSL-4 conditions.

  • JC virus quasispecies analysis reveals complex viral population underlying PML and supports viral dissemination via the hematogenous route [PublishAheadOfPrint]

  • Opportunistic infection of oligodendrocytes by human JC polyomavirus may result into development of progressive multifocal encephalopathy in immune compromised individuals. Neurotropic JC virus generally harbors reorganized non-coding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma and CSF) from nineteen PML patients, we attempted to reveal the composition of the JC polyomavirus population (the quasispecies i.e. the whole of the consensus and minor viral variants) contained in different body compartments and to better understand intra-patient viral dissemination. Our data demonstrate that in CSF of PML patients the JC viral population often is a complex mixture composed of multiple viral variants that contribute to the quasispecies. In contrast, urinary JC virus highly resembled archetype virus and most often did not contain minor viral variants. It also appeared that archetype JC virus sporadically can be identified in PML brain although selection of rearranged JC virus DNA is favored. Comparison of the quasispecies between different body compartments within a given patient suggested a strong correlation between the viral population in plasma and CSF whereas the viral population shed in urine appeared as unrelated. In conclusion, it is shown that the representation of viral DNA in the CSF following high level DNA replication in the brain underlying PML has hitherto been much underestimated. Our data also underscore that the hematogenous route might play a pivotal role in viral dissemination from or towards the brain.

    IMPORTANCE For the first time the JC polyomavirus population contained in different body compartments of patients diagnosed with progressive multifocal encephalopathy has been studied by deep sequencing. Two main findings came out of this work. First, it became apparent that the complexity of the viral population associated with PML has been highly underestimated so far, suggestive for a highly dynamic process of reorganization of the non-coding control region of JC polyomavirus in vivo, mainly in CSF and blood borne virus. Secondly, evidence was provided showing viral dissemination from and/or towards the brain via the hematogenous route, confirming a hypothesis that recently had been put forward in the field.

  • The murine norovirus (MNV) core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis [PublishAheadOfPrint]

  • All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein that functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3rrsquo; of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a non-coding context as it was possible to restore the replication of a MNV mutant by introducing an additional copy of the stem-loop between the NS7 and VP1 coding regions. Finally, in vitro biochemical data would suggest that stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RdRp, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.

    Importance Noroviruses are a significant cause of viral gastroenteritis and it is important to understand the mechanism of norovirus RNA synthesis. Herein we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA dependent RNA polymerase.

  • Nature of the N-terminal Amino Acid Residue of HIV-1 RNase H is Critical for the Stability of Reverse Transcriptase in Viral Particles [PublishAheadOfPrint]

  • Reverse transcriptase (RT) of Human Immunodeficiency Virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However unlike other viral proteins RT is subject to an internal cleavage event leading to the formation of two subunits in the virion nndash; a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H as an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway that is an ubiquitin dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to a structurally different amino acid than the conserved residue leads to degradation of RT and in some cases integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays we show that degradation of RNase H N-terminal mutant RT occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits.

    Importance HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein RNase H that is cleaved from reverse transcriptase. In particular we found that the first amino acid of RNase H never varies in over 1850 isolates of HIV-1 that we compared. When we changed the first amino acid we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins we show here that this is may not be the case for our mutants. Our results suggest that presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants suggesting a role for a cellular protease in this process.

  • Attenuated West Nile virus mutant NS1130-132QQA/175A/207A exhibits virus-induced ultrastructural changes and accumulation of protein in the Endoplasmic Reticulum [PublishAheadOfPrint]

  • We have previously shown that ablation of the three N-linked glycosylation sites in the West Nile virus NS1 protein completely attenuates mouse neuroinvasiveness (gge;1,000,000 PFU). Here we compared replication of the NS1130-132QQA/175A/207A mutant to parental NY99 strain in monkey kidney Vero cells. The results suggest that the mechanism of attenuation is due to lack of NS1 glycosylation, which blocks efficient replication, maturation and NS1 secretion from the ER, and results in changes of virus-induced ultrastructure.

  • Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus [PublishAheadOfPrint]

  • Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, Massachusetts, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein, which was demonstrated to have fusion activity, and also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated as VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined.

    Importance The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

  • Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodelling complex [PublishAheadOfPrint]

  • Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G and L), the 13.2 kb (-) ssRNA WONV genome contains five uncharacterised accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3) and a fifth overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-aa C-terminal domain of SNF5, a component of the SWI/SNF chromatin-remodelling complex. The interaction was confirmed by affinity chromatography and nuclear co-localisation was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are up-regulated during infection in mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knock-down results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine CSF1 gene. The data suggests that WONV U3 targets the SWI/SNF complex to block the host response to infection.

    Importance The rhabdoviruses comprise a large family of RNA viruses, infecting plants, vertebrates and invertebrates. In addition to the major structural proteins (N, P, M, G and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodelling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine CSF-1, suggesting that it targets the chromatin remodelling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression.

  • Eilat Virus Host Range Restriction is Present at Multiple Levels of Virus Life Cycle [PublishAheadOfPrint]

  • Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described and in contrast to all other mosquito-borne viruses is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3rrsquo; untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV non-structural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both non-structural (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses.

    Importance: Our work explores the nature of host range restriction of the first "mosquito-only alphavirus", EILV. EILV is related to pathogenic mosquito-borne viruses [eastern (EEEV), western WEEV), Venezuelan equine encephalitis (VEEV) and chikungunya (CHIKV)] that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission.

  • Less is More: Ebola Surface Glycoprotein Expression Levels Regulate Virus Production and Infectivity [PublishAheadOfPrint]

  • The Ebola virus surface glycoprotein (GP1,2) mediates host cell attachment and fusion, and is the primary target for host neutralizing antibodies. Expression of GP1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP1,2 expression impair production and release of EBOV VLPs, as well as infectivity of GP1,2-pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP1,2 containing viruses. Taken together, our findings indicate that GP1,2 expression levels have a profound effect on factors that contribute to virus fitness, and that RNA editing may be an important mechanism employed by EBOV to regulate GP1,2 expression in order to optimize virus production and infectivity.

    IMPORTANCE The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal with up to 90% mortality. The EBOV surface glycoprotein (GP1,2) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA editing mechanism during virus replication. Our study demonstrates that the level of GP1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.

  • Distinct endometrial and decidual macrophage characteristics and regulation of their permissivity to HIV-1 infection by SAMHD1 [PublishAheadOfPrint]

  • In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae, and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages (dM) are the main HIV-1 target cells in the decidua. Here we report that, in vitro, macrophages (eM) and T cells are the main HIV-1 targets in the non pregnant endometrium. As reported for dM, eM were found to have an M2-like phenotype (CD68+/CD163+/CD206+/IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of pro- and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role of SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with non pregnant FRT mucosae should help to identify mechanisms of mucosal protection against HIV-1 infection.

    Importance The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the non pregnant endometrium could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium, and that infection of macrophages from both the endometrium and the decidua is restricted by SAMHD1. These findings might have implications for the development of vaccines to prevent HIV-1 mucosal transmission.

  • A lethal murine infection model for dengue virus 3 in AG129 mice deficient in Type I and II interferon receptors leads to systemic disease [PublishAheadOfPrint]

  • The mosquito-borne disease dengue (DEN) is caused by four serologically and genetically related viruses, termed DENV-1 to DENV-4. Infection with one DENV usually leads to acute illness and results in lifelong homotypic immunity, but individuals remain susceptible to infection by the other three DENVs. The lack of a small animal model that mimics systemic DEN disease without neurovirulence has been an obstacle, but DENV-2 models have been recently developed in AG129 mice (deficient in interferon aalpha;/bbeta; and receptor signaling) that resemble human disease. However, comparable DENV-1, -3 and -4 models have not been developed. We utilized a non-mouse adapted DENV-3 Thai human isolate to develop a lethal infection model in AG129 mice. Intraperitoneal inoculation of six-to-eight-week old animals with strain C0360/94 led to rapid, fatal disease. Lethal C0360-94 infection resulted in physical signs of illness, high viral loads in the spleen, liver, and large intestine, histological changes in the liver and spleen tissues, and increased serum cytokine levels. Importantly, the animals developed vascular leakage, thrombocytopenia, and leukopenia. Overall, we have developed a lethal DENV-3 murine infection model, with no evidence of neurotropic disease based on a non-mouse adapted human isolate, which can be used to investigate DEN pathogenesis and to evaluate candidate vaccines and antivirals. This suggests that murine models utilizing non-mouse adapted isolates can be obtained for all four DENVs.

    Importance Dengue (DEN) is a mosquito-borne disease caused by four DENV serotypes (DENV-1, -2 -3 and -4) that have no treatments or vaccines. Primary infection with one DENV usually leads to acute illness followed by lifelong homotypic immunity, but susceptibility to infection by the other three DENVs remains. Therefore, a vaccine needs to protect from all four DENVs simultaneously. To date a suitable animal model to mimic systemic human illness only exists for DENV-2 in immunocompromised mice using passaged viruses; however, models are still needed for the remaining serotypes. This study describes establishment of a lethal systemic DENV-3 infection model with a human isolate in immunocompromised mice and is the first report of lethal infection by a non-adapted clinical DENV isolate without evidence of neurological disease. Our DENV-3 model provides a relevant platform to test DEN vaccines and antivirals.

  • Replication of Boid Inclusion Body Disease Associated Arenaviruses is Temperature Sensitive in Both Boid and Mammalian Cells [PublishAheadOfPrint]

  • Boid inclusion body disease (BIDB) is a fatal disease of boid snakes, the etiology of which has only recently been revealed following identification of several novel arenaviruses in diseased snakes. BIBD-associated arenaviruses (BIBDAV) are genetically divergent from the "classical" Old and New World arenaviruses and also differ substantially from each other. Even though there is convincing evidence that BIBDAV are indeed the etiological agent of BIBD, the BIBDAV reservoir hosts nndash; if any exist besides boid snakes themselves nndash; are not yet known. In this report we use University of Helsinki virus (UHV) to show that BIBDAV can effectively replicate also in mammalian, including human cells provided they are cultured at 30ddeg;C. The infection induces the formation of cytoplasmic inclusion bodies (IB), comprised mainly of viral nucleoprotein (NP), similar to those observed in BIBD and in boid cell cultures. Transfer of infected cells from 30ddeg;C to 37ddeg;C ambient temperature resulted in a progressive decline in IB formation and in the amount of viral NP and RNA, suggesting that BIBDAV growth is limited at 37ddeg;C. These observations indirectly indicate that IB formation is linked to viral replication. In addition to mammalian and reptilian cells, UHV infected arthropod (tick) cells when grown at 30ddeg;C. Even though our findings suggest that BIBDAV have a high potential to cross the species barrier, their inefficient growth at mammalian body temperatures indicates that the reservoir hosts of BIBDAV are likely species with a lower body temperature such as snakes.

    IMPORTANCE The newly-discovered boid inclusion body disease-associated arenaviruses (BIBDAV) of reptiles have drastically altered the phylogeny of the family Arenavirus. Prior to their discovery, known arenaviruses were considered as mainly rodent-borne viruses, with each arenavirus species having its own reservoir host. BIBDAV have so far been demonstrated in captive boid snakes but their possible reservoir host(s) have not yet been identified. Here we show, using University of Helsinki virus as a model, that these viruses are able to infect mammalian (including human) and arthropod cells. Our results provide in vitro proof of the considerable ability of arenaviruses to cross species barrier. However, our data indicate BIBDAV growth occurs at 30ddeg;C but is inhibited at 37ddeg;C, implying that crossing of the species barrier would be hindered by the body temperature of mammalian species.

  • Expression of the epidermodysplasia verruciformis-associated genes EVER1 and EVER2 is activated by exogenous DNA and inhibited by LMP1 oncoprotein from Epstein--Barr virus [PublishAheadOfPrint]

  • EVER1 and EVER2 are mutated in epidermodysplasia verruciformis patients, who are susceptible to beta HPV infection. It is unknown whether their products control the infection of other viruses. Here, we show that the expression of both genes in B cells is activated immediately after EBV infection, whereas at later stages it is strongly repressed via activation of NF-B signaling pathway by LMP1. Ectopic expression of EVER1 impairs the ability of EBV to infect B cells.

  • Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV Envelope [PublishAheadOfPrint]

  • The high-mannose patch of HIV envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently and consequently this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due to the quaternary nature of the bnAb epitopes and variability in neighboring loops and glycans critical to the epitopes. BnAbs against this region have some shared and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120 and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers some insight into these differences compared to PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described.

    Importance Development of an HIV vaccine is of vital importance for control of new infections and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from donor 36 (PGTs125-131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAb that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.

  • Three-dimensional structure of a protozoal dsRNA virus that infects enteric pathogen Giardia lamblia [PublishAheadOfPrint]

  • Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented dsRNA virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 AAring;. Its outermost diameter is 485 AAring;, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and spread through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans.

    IMPORTANCE Numerous pathogenic bacteria including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

  • Mutations in HIV-1 envelope that enhance entry with the macaque CD4 receptor alter antibody recognition by disrupting quaternary interactions within the trimer [PublishAheadOfPrint]

  • Chimeric SIV/HIV (SHIV) infection of macaques is commonly used to model HIV-1 transmission and pathogenesis in humans. Despite the fact that SHIVs encode SIV antagonists of the known macaque host restriction factors, these viruses require additional adaptation for replication in macaques to establish a persistent infection. Additional adaptation may be required in part because macaque CD4 (mCD4) is a suboptimal receptor for most HIV-1 Env variants. This requirement raises the possibility that adaptation of HIV-1 Env to the macaque host leads to selection of variants that lack important biological and antigenic properties of the viruses responsible for the HIV-1 pandemic in humans. Here, we investigated whether this adaptation process leads to changes in the antigenicity and structure of HIV-1 Env. For this purpose, we examined how two independent mutations that enhance mCD4-mediated entry, A204E and G312V, impact antibody recognition in the context of seven different parental HIV-1 Envs from diverse subtypes. We also examined HIV-1 Env variants from three SHIVs that had been adapted for increased replication in macaques. Our results indicate that these different macaque-adapted variants shared common features including resistance to antibodies directed to quaternary epitopes and sensitivity to antibodies directed to epitopes in the variable domains (V2 and V3) that are buried in the parental, un-adapted Envs. Collectively, these findings suggest that adaptation to mCD4 results in conformational changes that expose epitopes in the variable domains and disrupt quaternary epitopes in the native Env trimer.

    Importance: These findings indicate the antigenic consequences of adapting HIV-1 Env to macaque CD4. They also suggest that to best mimic HIV-1 infection in humans using the SHIV/macaque model, HIV-1 Envs should be identified that use mCD4 as a functional receptor and preserve quaternary epitopes characteristic of HIV-1 Env.

  • Activation of the chicken type I IFN response by infectious bronchitis coronavirus. [PublishAheadOfPrint]

  • Coronaviruses from both the Alpha and Betacoronavirus genera, interfere with the type I interferon (IFN) response in various ways, ensuring limited activation of the IFN response in most cell types. Of Gammacoronaviruses that mainly infect birds, little is known about activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells and in a chicken cell line. Ifnbbeta; expression in fact, is delayed with respect to the peak of viral replication and accompanying accumulation of dsRNA. In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the IFN response at the transcriptional and translational level. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, signalling of non-self dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.

    Importance: Our results demonstrate that IBV has evolved multiple strategies to avoid activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction, and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as pathogenesis of Gammacoronaviruses.

  • Complete genome characterization of recent and ancient Belgian pig group A rotaviruses and assessment of their evolutionary relationship with human rotaviruses [PublishAheadOfPrint]

  • Group A rotaviruses (RVAs) are an important cause of diarrhea in young pigs and children. An evolutionary relationship has been suggested to exist between pig and human RVAs. This hypothesis was further investigated by phylogenetic analysis of the complete genomes of six recent (G2P[27], G3P[6], G4P[7], G5P[7], G9P[13], and G9P[23]) and one historic (G1P[7]) Belgian pig RVA strains, and of all completely characterized pig RVAs around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4 and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2 and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1 and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7 further confirm this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6]-strains, but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted.

    Importance Rotaviruses are an important cause of diarrhea in many species including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data of pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced and their evolutionary relationship with human Wa-like strains was investigated. Our data show that Wa-like human and pig strains have entered different evolutionary paths. Our data indicate that pig P[6]-strains form the most considerable risk for interspecies transmission to humans. However, efficient spread of pig strains in the human population is most likely hampered by the adaptation of some crucial viral proteins to the cellular machinery of pig enterocytes. These data allow a better understanding of the risk for direct interspecies transmission events and the emergence of pig rotaviruses or pig-human reassortants in the human population.

  • Prevention of Measles Infection by intranasal delivery of fusion inhibitory peptides [PublishAheadOfPrint]

  • Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting into the target cell membrane and then re-folding into a post-fusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated, and helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide, and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immune compromised hosts.

    Importance Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small animal models of MV infection, that fusion inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside of health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

  • Short Conserved Sequences of HIV-1 Are Highly Immunogenic and Shift Immunodominance [PublishAheadOfPrint]

  • Cellular immunity is pivotal in HIV-1 pathogenesis, but hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore we selected four relatively conserved regions (Gag amino acids 148-214 and 250-335, Env 521-606, and Nef 106-148), each created in three mosaics to provide better coverage of M-group HIV-1 sequences. A conserved region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics (each region at a separate injection site) was compared to a whole protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus-5 boost (weeks 0, 4, 8, and 24 respectively). Although CRV inserts were about a fifth that of WPV, the CRV generated comparable magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions versus the outside regions. The CRV yielded conserved region targeting density that was approximately five-fold that of the WPV. A similar pattern was seen in bronchoalveolar lymphocytes, but quadruple the magnitudes in blood. Overall, these findings demonstrated that the selected conserved regions are highly immunogenic, and that anatomically isolated vaccinations with these regions focuses immunodominance compared to full-length protein vaccination.

    IMPORTANCE HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one fifth of whole Gag/Env/Nef, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate immunogenicity of these small conserved regions, and the potential for a vaccine to steer immunodominance towards conserved epitopes.

  • White Spot Syndrome Virus Protein Kinase 1 (PK1) Defeats the Host Cell's Iron Withholding Defense Mechanism by Interacting with Host Ferritin [PublishAheadOfPrint]

  • Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin by the yeast two-hybrid system. A pull-down assay and 27-MHz quartz crystal microbalance (QCM) confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron withholding defense mechanism.

    Importance Statement We show here that the white spot syndrome virus ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counter-strategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and the host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, maintains the intracellular labile iron pool (LIP) - and thus the availability of free iron - is increased within cells.

  • Class switch recombination and somatic hypermutation of virus- neutralizing antibodies are not essential for the control of Friend retrovirus infection [PublishAheadOfPrint]

  • Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient of activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4+ T cells.

  • The influenza NA protein transmembrane and head domains have co-evolved [PublishAheadOfPrint]

  • Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical llsquo;oldrrsquo; TMD (1933) with a llsquo;recentrrsquo; (2009) more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37ddeg;C compared to 33ddeg;C where NA folds more efficiently. Passaging the chimera viruses at 37ddeg;C restored the NA folding efficiency, viral budding and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggests the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain.

    IMPORTANCE The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an llsquo;oldrrsquo; N1 head domain from 1933 is incompatible with a llsquo;recentrrsquo; (2009) more polar N1 TMD sequence, and that during viral replication the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have co-evolved.

  • A conserved regulatory module at the C-terminus of the papillomavirus E1 helicase domain controls E1 helicase assembly [PublishAheadOfPrint]

  • Viruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double stranded ori DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.

    Importance. This study provides a mechanistic understanding into how a multi-functional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+ helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

  • Cognate CD4 T-cell licensing of dendritic cells heralds anti-CMV CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation [PublishAheadOfPrint]

  • Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord-blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells is required to generate effective CD8+ T-cell responses. For humans this was not fully understood. We here show that CD4+ T-cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65 specific CD4+ T-cells precedes the rise in CMV-pp65 specific CD8+ T-cells. Second, the elicitation of CMV-pp65 specific CD8+ T-cells from rare naiiuml;ve precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T-cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of IFN- by pp65-specific CD4+ T-cells. Together these data show that human DCs require the licensing by cognate antigen-specific CD4+ T-cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients.

    Importance paragraph: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T-cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T-cells, called helper T-cells, is required.

  • Rotavirus entry, a deep journey into the cell with several exits [PublishAheadOfPrint]

  • Rotaviruses are the leading etiological agents of acute gastroenteritis in infants and young children worldwide. These nonenveloped viruses enter cells using different types of endocytosis and, depending on the virus strain, travel to different endosomal compartments before exiting to the cytosolic space. In this GEM we review the viral and cellular factors involved in the different stages of a productive virus cell entry and share with the readers the journey we have taken into the cell to learn about virus entry.

  • Chronic administration of {Delta}9-tetrahydrocannabinol induces intestinal anti-inflammatory microRNA expression during acute SIV infection of rhesus macaques [PublishAheadOfPrint]

  • Recreational and medical use of cannabis among HIV-infected individuals has increased in recent years. In SIV-infected macaques, chronic administration of 9-tetrahydrocannabinol (9-THC), inhibited viral replication, intestinal inflammation and slowed disease progression. Persistent gastrointestinal disease/inflammation has been proposed to facilitate microbial translocation, systemic immune activation and promote disease progression. Cannabinoids including 9-THC attenuated intestinal inflammation in mouse colitis models and SIV-infected rhesus macaques. To determine if the anti-inflammatory effects of 9-THC involved differential microRNA (miRNA) modulation, we profiled miRNA expression at 14, 30 and 60 days post-infection (DPI) in the intestine of uninfected macaques receiving 9-THC (n=3) and SIV-infected macaques administered either vehicle (VEH/SIV; n=4) or THC (THC/SIV; n=4). Chronic 9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60DPI. At 60DPI, ~28% of miRNAs showed decreased expression in VEH/SIV compared to none in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149 and miR-187 previously been shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator was confirmed as a direct miR-99b target. A significant increase in NOX4+ crypt epithelial cells was detected in VEH/SIV compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. These results support a role for differential miRNA induction in THC-mediated suppression of intestinal inflammation. Whether similar miRNA modulation occurs in other tissues requires further investigation.

    IMPORTANCE Gastrointestinal tract (GI) disease/inflammation is a hallmark of HIV/SIV infection. Previously, we showed that chronic treatment of SIV-infected macaques with 9 tetrahydrocannabinol (9-THC) increased survival and decreased viral replication and infection induced gastrointestinal inflammation. Here, we show that chronic THC administration to SIV-infected macaques induced an anti-inflammatory microRNA expression profile in the intestine at 60DPI. These included several miRNAs bioinformatically predicted to directly target CXCL12, a chemokine known to regulate lymphocyte and macrophage trafficking into the intestine. Specifically, miR-99b was significantly upregulated in THC-treated SIV-infected macaques and confirmed to directly target NADPH oxidase 4 (NOX4), a reactive oxygen species generator known to damage intestinal epithelial cells. Elevated miR-99b expression was associated with a significantly decreased number of NOX4+ epithelial cells in the intestines of THC-treated SIV-infected macaques. Overall, our results show that selective upregulation of anti-inflammatory miRNA expression, contributes to THC-mediated suppression of gastrointestinal inflammation and maintenance of intestinal homeostasis.

  • miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a [PublishAheadOfPrint]

  • Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs and induces cell-cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products, IE1, pp71, and UL26, were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels post HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.

    IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus/host interactions that govern (HCMV) replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection down regulates miR-21, but up regulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increase of Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic anti-viral factor that represents a potential target for therapeutic intervention.

  • Evidence of lymphocytic choriomeningitis virus in domestic mice in Gabon; risk of emergence of LCMV encephalitis in Central Africa [PublishAheadOfPrint]

  • Lymphocytic choriomeningitis virus (LCMV) can cause acute fatal disease in all continents but was never detected in Africa. We report the first detection of LCMV RNA in common European house mouse (Mus musculus domesticus) in Africa. Phylogenetic analyses showed close relationship with North American strains. These findings suggest appearance of LCMV acute encephalitis cases. This is a perfect example of virus dissemination by its natural host that may have dramatic public health consequences.

  • IMPACT AND REGULATION OF LAMBDA INTERFERON RESPONSE IN HUMAN METAPNEUMOVIRUS INFECTION [PublishAheadOfPrint]

  • Human metapneumovirus (hMPV) is a worldwide distributed respiratory paramyxovirus that induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response against this virus are still largely unknown. In this study we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce the expression on IFN-1, IFN-2, IFN-3 and IFN-4. The induction of IFN- by hMPV was dependent of IRF-7, but not IRF-3, expression. Treatment of hMPV-infected mice with IFN- reduced disease severity, lung viral titer and inflammatory response in the lung. Moreover, the IFN- response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-s) play a critical protective role in hMPV infection.

    Importance Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs) constitute an indispensable part of the innate immune response including IFN-, the newest addition to the interferon family. Here, we demonstrate that IFN- exhibits a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

  • Conformational Changes in Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment [PublishAheadOfPrint]

  • Virus capsids provide genome protection from environmental challenges, but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to the hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH, but undergoes more subtle conformational changes. These are indicated by an increased sensitivity to SDS-mediated dissociation and to dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding of low pH-treated hexon. Dispase cleavage, which we find maps to a specific site within the hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry, but inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and strongly inhibited in the post-entry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus.

    IMPORTANCE The adenovirus 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport towards the nucleus and AdV5 infectivity. In this work, we describe how low pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1 which depends on the same structural changes and, furthermore, regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating the long known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

  • Efficient infectious cell culture systems of the hepatitis C virus prototype strains HCV-1 and H77 [PublishAheadOfPrint]

  • The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell-culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5rrsquo; UTR-NS5A and the JFH1 NS5B-3rrsquo; UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. Combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with seventeen mutations (HCV1cc) replicated efficiently in Huh7.5 cells, and produced supernatant infectivity titers of 104.0 focus-forming-units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R identified here from culture-adapted full-length TN-viruses and a common NS3-helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants initiated replication and culture-adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harbouring nineteen mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naiiuml;ve Huh7.5 cells, reaching titers of 103.5 and 104.4 FFU/ml, respectively.

    Importance Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell-culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a(TN), 2a(J6), and 2b(J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

  • Co-circulation of two distinct genetic and antigenic lineages of proposed influenza D virus in cattle [PublishAheadOfPrint]

  • Viruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real time reverse transcription PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolated in vitro. Phylogenetic analysis of full genome sequences of these six new viruses and four previously reported viruses revealed two distinct co-circulating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660) which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross reactivity against heterologous clade antiserum. One isolate, D/bovine/Kansas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and at least two genetic and antigenically distinct clades co-circulate.

    Importance: A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with prevalence similar to other established BRDC etiological agents. These results are in good agreement with near ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct co-circulating lineages of IDV which freely reassort. Significant antigenic differences were observed between the two lineages that generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny. These results, in addition to the ability of IDV to infect and transmit in multiple mammalian species, warrant additional studies to determine the pathogenic potential of IDV.

  • Simian Hemorrhagic Fever Virus Cell Entry is Dependent on CD163 and Uses a Clathrin-mediated Endocytosis-like Pathway [PublishAheadOfPrint]

  • Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques, but is thought to be nonpathogenic for humans. To date, the SHFV lifecycle is almost completely uncharacterized on the molecular level. Here we describe the first steps of the SHFV lifecycle. Our experiments indicate that SHFV enters target cells by low pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-bbeta;-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin, and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knock-out study and electron-microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, aalpha;-chymotrypsin, trypsin) abrogated entry, indicating that the SHFV cell-surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component.

    IMPORTANCE Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa viruses. However, little is known about SHFV's ecology, molecular biology, and the mechanism by which it causes disease. Results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low pH-dependent, actin-independent endocytosis, with the likely help of a cellular surface protein.

  • T cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr virus [PublishAheadOfPrint]

  • Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells but the response has only been characterized in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating "immune blind spots" through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy.

    Importance Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by greatly expanding the number of defined T cell epitopes. An unusual clustering of epitopes was identified, highlighting a small region of BZLF1 that is targeted by the immune response of a high proportion of the world's population. This focussing of the immune response could be utilized in developing vaccines/therapies with wide coverage, or it could potentially be exploited by the virus to escape the immune response.

  • The Megavirus chilensis Cu,Zn-Superoxide Dismutase: the first viral structure of a typical CCS-independent hyperstable dimeric enzyme [PublishAheadOfPrint]

  • Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of Mimivirus purified virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper-zinc superoxide dismutase (SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions were required for the formation of the active site lid and dimer stabilization, M. chilensis SOD forms a very stable metal-free dimer. We used EPR analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 was sufficient to restore a fully active holo enzyme. These results demonstrate that the viral enzyme activation is independent of a chaperone both for disulfide bridge formation and copper incorporation, and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from Mimivirus. As in Poxviruses, the enzyme might be dispensable when infecting Acanthamoeba cells but could allow M. chilensis to infect a broader range of eukaryotic hosts.

    Importance Mimiviridae are giant viruses encoding more than 1000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole oxidative stress. Megavirus chilensis virion contain a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some Megavirus chilensis relatives but is absent from Mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the common features and differences of the viral enzyme compared to cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon growth medium supplementation of Cu and Zn the recombinant protein is fully active, suggesting that the viral SOD activation is independent on a copper chaperone for SOD generally used by eukaryotic SODs.

  • Enhanced transgene expression from single-stranded D-sequence-substituted recombinant AAV vectors in human cell lines in vitro and in murine hepatocytes in vivo [PublishAheadOfPrint]

  • We have previously reported that the removal of a 20-nucleotide sequence, termed the D-sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome, significantly impairs rescue, replication, and encapsidation of the viral genomes (J. Mol. Biol., 250: 573-580, 1995; J. Virol., 70: 1668-1677, 1996). Here we describe that substitution of only one D-sequence in either ITR restores each of these functions, but DNA strands of only single-polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded (ss) DNA, which is transcriptionally-inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription-suppressor sequence in one of the D-sequences, which shares homology with the binding site for the cellular NF-B-repressing factor (NRF). The removal of this D-sequence from, and substitution with a sequence containing putative binding sites for transcription factors in, ssAAV vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only in the basic biology of AAV, but also in the optimal use of these vectors in human gene therapy.

    Importance The results of the studies described here have provided not only novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV) that causes no known disease, but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased level of gene expression. Thus, these studies have significant implications in the use of these novel AAV vectors in their potential use in human gene therapy.

  • High-Throughput RNA sequencing based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project [PublishAheadOfPrint]

  • Using high-throughput RNA sequencing (RNA-seq) data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of MuLV transcripts was observed in DEL cells and we identified 4 transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes including IL15, IL6ST, STAT5B, HIVEP1, and IL9R. While KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL6, K4/vIL8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen, LANA. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.

    Importance Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus 1 (HTLV-1) are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes including cytokine regulators that are disrupted by virus integration and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

  • Varicella-zoster virus-derived MHC class I-restricted peptide affinity is a determining factor for HLA risk profile to develop PHN [PublishAheadOfPrint]

  • Postherpetic neuralgia (PHN) is the most common complication of herpes zoster and is typified by a lingering pain that can last months or years after the characteristic herpes zoster rash disappears. It is well known that there are risk factors for the development of PHN, such as its association with certain HLA alleles. In this study, previous HLA-genotyping results were collected and subjected to a meta-analysis with increased statistical power. This work shows that the alleles HLA-A*33 and HLA-B*44 are significantly enriched in PHN patients, while HLA-A*02 and HLA-B*40 are significantly depleted. Prediction of the varicella-zoster virus (VZV) peptide affinity for these four HLA variants using two existing and one in-house-developed state-of-the-art MHC class I ligand prediction methods reveals that there is a great difference in their absolute and relative peptide binding repertoires. It was observed that HLA-A*02 displays a high affinity for approximately seven-fold higher number of VZV peptides than HLA-B*44. Further, after correction for HLA-allele specific limitations, the relative affinity for VZV peptides of HLA-A*33 and HLA-B*44 was found to be significantly less than those of HLA-A*02 and HLA-B*40. In addition, HLA peptide affinity calculations indicate strong trends for VZV to avoid high affinity peptides in some of its proteins, independent from the studied HLA allele.

    Importance The varicella-zoster virus can cause two distinct diseases: chickenpox (varicella) and shingles (herpes zoster). Varicella is a common disease in young children, while herpes zoster is more frequent in older individuals. A common complication of herpes zoster is postherpetic neuralgia, a persistent and debilitating pain that can remain months up to years after the resolution of the rash. In this study, we show that the affinity for varicella-zoster peptides of HLA variants associated with higher postherpetic neuralgia risk have a lower relative affinity for varicella-zoster peptides than variants with a lower risk. These results provide new insight into the development of postherpetic neuralgia and strongly support the hypothesis that one of its possible underlying causes is a suboptimal anti-VZV immune response due to weak HLA-binding peptide affinity.

  • Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs [PublishAheadOfPrint]

  • The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation and vaccine regimen. In this study, we have generated two novel NYVAC vectors expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef) and defined their virological and immunological characteristics in cultured cells and in mice. Insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells, Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights in virus-host cell interactions of NYVAC vectors expressing HIV antigens, with activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors.

    Importance We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) or Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable, express high levels of both HIV-1 antigens, Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered as vaccine candidates against HIV/AIDS and are currently being tested in macaques and humans.

  • Uneven genetic robustness of HIV-1 integrase [PublishAheadOfPrint]

  • Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single amino acid mutants in HIV-1 integrase (IN), covering ggt;40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random non-silent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing and particle-associated IN levels were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates overall robustness of IN, and highlights particular regions of vulnerability that may be targeted in therapeutic interventions.

    Importance The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single amino acid IN mutations that could mimic the types of mutations that naturally occur during HIV-1 infection. Previously, we measured the robustness of HIV-1 capsid in this manner and determined that it is extremely intolerant of mutation. In contrast to CA, HIV-1 IN proved relatively robust, with far fewer mutations causing lethal defects. However, when we subsequently mapped the lethal mutations onto a model of the structure of the multi-subunit IN-viral DNA complex, we found the lethal mutations that caused virus morphogenesis defects tended to be highly localized at subunit interfaces. This discovery of vulnerable regions of HIV-1 IN could inform development of novel therapeutics.

  • The Hepatitis B Virus HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival [PublishAheadOfPrint]

  • Chronic infection with the hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor Hepatocyte Nuclear Factor (HNF) 4aalpha; is a target of HBx-regulated AKT and link HNF4aalpha; to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication.

    Importance A chronic hepatitis B virus (HBV) infection is a common cause for the development of liver cancer. Regulation of cell-signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an essential role of HBx during HBV replication, HBx activation of AKT inhibits hepatocyte apoptosis, and this may facilitate persistent, noncytopathic HBV replication. AKT regulates HBV replication by reducing the activity of the transcription factor Hepatocyte Nuclear Factor (HNF) 4aalpha;. HBx activation of AKT may contribute to the development of liver cancer by facilitating persistent HBV replication, augmenting dedifferentiation of hepatocytes by inhibiting HNF4aalpha; functions, and activating AKT-regulated oncogenic pathways. AKT-regulated factors may provide therapeutic targets for inhibiting HBV replication and the development of HBV-associated liver cancer.

  • Modulation of Type I interferon-associated viral sensing during acute simian immunodefiency virus (SIV) infection in African green monkeys [PublishAheadOfPrint]

  • Natural hosts of simian immunodeficiency virus (SIV), such as African green monkeys (AGMs), do not progress to AIDS. This associates with an absence of a chronic type I interferon signature. It is unclear how the IFN-I response is downmodulated in AGMs. We longitudinally assessed the capacity of AGM blood cells to produce IFN-I in response to SIV and Herpes Simplex virus (HSV). Phenotype and function of plasmacytoid dendritic cells (pDCs) and other mononuclear blood cells were assessed by flow cytometry and expression of viral sensors measured by RT-PCR. pDC displayed low BDCA-2, CD40 and HLA-DR expression during acute SIVagm infection. BDCA-2 was required for sensing SIV, but not HSV, by pDCs. In acute infection, AGM PBMCs produced less IFN-I upon SIV stimulation. In chronic phase the production was normal, confirming that the lack of chronic inflammation is not due to a sensing defect of pDCs. In contrast to stimulation by SIV, more IFN-I was produced upon HSV stimulation of PBMC isolated during acute infection, while the frequency of AGM pDCs producing IFN-I upon in vitro stimulation with HSV was diminished. Indeed, other cells started producing IFN-I. This increased viral sensing by non-pDCs was associated with an upregulation of TLR3 and IFI16 caused by IFN-I in acute SIVagm infection. Our results suggest that, as in pathogenic SIVmac infection, SIVagm infection mobilizes bone marrow precursor pDC. Moreover, we show that SIV infection modifies the capacity of viral sensing in cells other than pDCs which could drive IFN-I production in specific settings.

    Importance The effect of HIV/SIV infections on the capacity of plasmacytoid dendritic cells (pDC) to produce IFN-I in vivo is still incompletely defined. As IFN-I can restrict viral replication, contribute to inflammation and influence immune responses, alteration of this capacity could impact viral reservoir size. We observed that even in non-pathogenic SIV infection, the frequency of pDC capable to efficiently sense SIV and produce IFN-I is reduced during acute infection. We discovered that, concomitantly, cells other than pDCs increased their ability of viral sensing. Our results suggest that pDC-produced IFN-I upregulate viral sensors in bystander cells, the latter gaining the capacity to produce IFN-I. These results indicate that in certain settings, cells other than pDCs can drive IFN-I-associated inflammation in SIV infection. This has important implications for the understanding of persistent inflammation and the establishment of viral reservoirs.

  • A Novel Factor I Activity in Nipah Virus Inhibits Human Complement Pathways Through Cleavage of C3b [PublishAheadOfPrint]

  • Complement (C') is an innate immune system that most animal viruses must face during natural infections. Given that replication and dissemination of the highly pathogenic Nipah virus (NiV) includes exposure to environments rich in C' factors, we tested the in vitro sensitivity of NiV to C'-mediated neutralization. Here we show that NiV was completely resistant to in vitro neutralization by normal human serum (NHS). Treatment of purified NiV with NHS activated C' pathways, but there was very little C3 deposition on virus particles. In in vitro reconstitution experiments, NiV particles provided time- and dose-dependent Factor I-like protease activity capable of cleaving C3b into inactive iC3b. NiV-dependent inactivation of C3b only occurred with cofactors Factor H and soluble CR1, but not with CD46. Purified NiV particles did not support C4b cleavage. Electron microscopy of purified NiV particles showed immunogold-labeling with anti-Factor I antibodies. Our results suggest a novel mechanism by which NiV evades the human C' system through a unique Factor I-like activity.

    IMPORTANCE Viruses have evolved mechanisms to limit C'-mediated neutralization, some of which involve high-jacking cellular proteins involved in control of inappropriate C' activation. Here we report a previously unknown mechanism whereby NiV provides a novel protease activity capable of in vitro cleavage and inactivation of C3b, a key component of the C' cascade. These data help to explain how an enveloped virus such as NiV can infect and disseminate through body fluids that are rich in C' activity. Disrupting the ability of NiV to recruit C' inhibitors could form the basis for the development of effective therapies and safer vaccines to combat these highly pathogenic emerging viruses.

  • Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers [PublishAheadOfPrint]

  • Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate the viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of Dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer.

    IMPORTANCE The structure of a Dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of post-fusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130ddeg; to the post-fusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" post-fusion conformation, thus inhibiting fusion.

  • APOBEC3A Functions as a Restriction Factor of Human Papillomavirus [PublishAheadOfPrint]

  • Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers including cervical and head and neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head and neck cancers exhibited higher rates of hA3 mutation signatures compared to most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis.

    IMPORTANCE Human papillomaviruses (HPV) are highly prevalent and potent human pathogens that cause over 5% of all human cancers, including cervical and head and neck cancers. While the majority of people become infected with HPV, only 10-20% of infections are established as persistent infections. This suggests the existence of intrinsic host defense mechanisms that inhibit viral persistence. Using a robust method to produce infectious HPV virions, we demonstrate that hA3A, but not hA3B or hA3C, can significantly inhibit HPV infectivity. Moreover, hA3A and hA3B were coordinately induced in HPV-positive clinical specimens during cancer progression, likely through an HPV E7 oncoprotein-dependent mechanism. Interestingly, HPV-positive cervical and head and neck cancers were recently shown to harbor significant amounts of hA3 mutation signatures. Our findings raise the intriguing possibility that the induction of this host restriction mechanism by HPV may also trigger hA3A- and hA3B-induced cancer mutagenesis.

  • Shift in Monocyte Apoptosis with Increasing Viral Load and Change in Apoptosis-related ISG/Bcl2-family Gene Expression in Chronic HIV-1 Infected Subjects [PublishAheadOfPrint]

  • Although monocytes and macrophages are targets of HIV-1-mediated immunopathology, the impact of high viremia on activation-induced monocyte apoptosis relative to monocyte and macrophage activation changes remains undetermined. Here, we determined constitutive and oxidative stress-induced monocyte apoptosis in uninfected and HIV(+) individuals across a spectrum of viral load (n=35, range: 2,243 to 1,355,998 HIV-1 RNA copies/mL) and CD4 count (range: 26 to 801 cells/mm3). Both constitutive and oxidative stress-induced apoptosis were positively associated with viral load and negatively associated with CD4 with an elevation in apoptosis occurring in patients with greater than 40,000 (4.6 log) copies/mL. As expected, expression of Rb1 and interferon-stimulated genes (ISGs), plasma sCD163 concentration, and the proportion of CD14+/CD16+ intermediate monocytes were elevated in viremic patients compared to uninfected controls. Although most ISG expression, sCD14, sCD163, and CD14+/CD16+ frequencies were not directly associated with a change in apoptosis, sCD14 and ISG expression showed an association with increasing viral load. Multivariable analysis of clinical values and monocyte gene expression identified changes in IFI27, IFITM2, Rb1, and Bcl2 expression as determinants of constitutive apoptosis (p=3.77x10-5, adjusted-R2=0.5983), while changes in viral load, IFITM2, Rb1, and Bax expression were determinants of oxidative stress-induced apoptosis (p=5.59x10-5, adjusted-R2=0.5996). Our data demonstrate differential activation states in monocytes between levels of viremia in association with differences in apoptosis that may contribute to greater monocyte turnover with high viremia.

    IMPORTANCE This study characterizes differential monocyte activation, apoptosis, and apoptosis-related gene expression in low versus high level viremic HIV-1 patients, suggesting a shift in apoptosis regulation that may be associated with disease state. Using single and multivariable analysis of monocyte activation parameters and gene expression, we support the hypothesis that monocyte apoptosis in HIV disease is a reflection of viremia and activation state with contributions from gene expression changes within the ISG/Bcl2-gene families. Understanding monocyte apoptosis response may inform HIV immunopathogenesis, retention of infected macrophages, and monocyte turnover in low or high viral load states.

  • Polyreactivity and Autoreactivity among HIV-1 Antibodies [PublishAheadOfPrint]

  • It is generally acknowledged that human broadly neutralizing Abs (bNAbs) capable of neutralizing multiple HIV-1 clades are often poly- or autoreactive. Whereas poly-/autoreactivity has been proposed to be crucial for neutralization breadth, no systematic, quantitative study of self-reactivity among non-neutralizing HIV-1 Abs (nNAbs) has been performed to determine whether poly- or autoreactivity in bNAbs is a consequence of chronic antigen (Ag) exposure and/or inflammation or a fundamental property of neutralization. Here, we use protein microarrays to assess binding to ggt;9,000 human proteins and find that as a class, bNAbs are significantly more poly- and autoreactive than nNAbs. The poly- and autoreactive property is therefore not due to the infection milieu, but rather associated with neutralization. Our observations are consistent with a role of heteroligation for HIV-1 neutralization and/or structural mimicry of host Ags by conserved HIV-1 neutralization sites. Although bNAb are more mutated than nNAbs as a group, V(D)J mutation per se does not correlate with poly- and autoreactivity. Infrequent poly- or autoreactivity among nNAbs implies that their dominance in humoral responses is due to absence of negative control by immune regulation. Interestingly, four of nine bNAbs specific for the HIV-1 CD4 binding site (CD4bs) (VRC01, VRC02, CH106, and CH103) bind human ubiquitin ligase E3A (UBE3A) and UBE3A protein competitively inhibits gp120 binding to the VRC01 bNAb. Among these four bNAbs, avidity for UBE3A was correlated with neutralization breadth. Identification of UBE3A as a self-antigen recognized by CD4bs bNAbs offers a mechanism for the rarity of this bNAb class.

    IMPORTANCE Eliciting bNAb is key for HIV-1 vaccines; most Abs elicited by HIV-1 infection or immunization, however, are strain-specific or non-neutralizing, and unsuited for protection. Here, we compare the specificities of bNAbs and nNAbs to demonstrate that bNAbs are significantly more poly- and autoreactive than nNAbs. The strong association of poly- and autoreactivity with bNAbs, but not nNAbs from infected patients, indicates that the infection milieu, chronic inflammation and Ag exposure, CD4 T cell depletion, etc., alone does not cause poly- and autoreactivity. Instead, these properties are fundamentally linked to neutralization breadth, either by requirement for heteroligation or the consequence of host mimicry by HIV-1. Indeed, we show that human UBE3A shares an epitope(s) with HIV-1 envelope recognized by four CD4bs bNAbs. The poly- and autoreactivity of bNAbs surely contribute to the rarity of MPER and CD4bs bNAbs, and identify a roadblock that must be overcome to induce protective vaccines.

  • Regulation of Influenza A Virus Nucleoprotein Oligomerisation by Phosphorylation [PublishAheadOfPrint]

  • In the influenza virus ribonucleoprotein complex the oligomerisation of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighbouring molecule. In this study we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerisation and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerisation in infected cells is regulated by reversible phosphorylation.

  • A newly-isolated reovirus has the simplest genomic and structural organization of any reovirus [PublishAheadOfPrint]

  • A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segmented, dsRNA virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryoEM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events.

    Importance We describe the detection, genetic, phenotypic and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature, or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

  • Mutation V111I in HIV-2 reverse transcriptase increases the fitness of the nucleoside analogue resistant K65R and Q151M viruses [PublishAheadOfPrint]

  • Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with combination of nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies showed that mutations in the connection and RNaseH domain of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, we therefore analysed the polymerase, connection and RNaseH domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation V111I in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNaseH domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility, but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses, but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-115 loop region, which may affect dNTP binding and polymerase activity.

    Importance Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues, but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

  • Amplified and Persistent Immune Responses Generated by Single Cycle Replicating Adenovirus Vaccines [PublishAheadOfPrint]

  • Replication-competent adenoviral vectors (RC-Ad) generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common E1-deleted replication-defective Ad (RD-Ad) vectors avoid this risk, but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered "single cycle" adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome, but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad. Surprisingly, SC-Ad, but not RC-Ad generated higher transgene-specific antibody levels than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher IFN- responses and higher transgene-specific serum antibody levels. These data suggest SC-Ad vectors may have utility as mucosal vaccines.

    Importance This work illustrates the utility of our recently developed single cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows replicating SC-Ad6 produces higher levels of luciferase and induces higher GFP-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not elicit comparable anti-GFP antibodies in permissive hamsters. When tested in the larger rhesus macaque model, SC-Ad6 induces higher transgene-specific antibody and T cell responses. Together this data suggests SC-Ad6 could be a more effective platform for developing vaccines against more relevant antigens. This could be especially beneficial for developing vaccines for pathogens where traditional replication defective adenovirus vectors have not been effective.

  • CD4+ T cell help is dispensable for protective CD8+ T cell memory against mousepox following vaccinia virus immunization [PublishAheadOfPrint]

  • It has been shown in various infection models that CD4+ T cell help (TH) is necessary for the conditioning, maintenance and/or recall responses of memory CD8+ T cells (CD8M). Yet, in the case of vaccinia virus (VACV), which constitutes the vaccine used to eradicate smallpox and is a candidate vector for other infectious diseases, the issue is controversial because different groups have shown either TH dependence or independence of CD8M conditioning, maintenance and or recall response. In agreement with some of these groups, we show that TH plays a role but is not essential for the maintenance, proliferation and effector differentiation of polyclonal memory CD8+ T cells after infection with wild type VACV WR. More important, we show that un-helped and helped anti-VACV memory CD8+ T cells are similarly efficient at protecting susceptible mice from lethal mousepox, the mouse equivalent of human smallpox. Thus, TH is not essential for the conditioning and maintenance of memory CD8+ T cells capable of mounting a recall response strong enough to protect from a lethal natural pathogen. Our results may partly explain why the VACV vaccine is so effective.

    Importance We used vaccinia virus (VACV) -a gold standard vaccine - as the immunogen, and ectromelia virus (ECTV) as the pathogen to demonstrate that the conditioning and maintenance of anti-VACV memory CD8+ T cells and their ability to protect against an Orthopoxvirus (OPV) infection in its natural host can develop in the absence of CD4+ T cell help. Our results provide important insight to our basic knowledge of the immune system. Further, because VACV is used as a vaccine in humans, our results may help us understand how this vaccine induces protective immunity in this species. In addition, this work may partly explain why VACV is so effective as a vaccine.

  • Discovery and characterization of auxiliary proteins encoded by Simian T-cell Lymphotropic Viruses type 3 [PublishAheadOfPrint]

  • HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency and immune escape. The presence of auxiliary proteins-encoding ORFs in HTLV-3, the latest HTLV to be discovered is unknown. STLV-3 viruses are almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using RT-PCR, we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8 and 9 kDa, and confirmed the presence of the previously reported RorfII transcript. The existence of those viral mRNAs was confirmed using splice site-specific RT-PCR in ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in calreticulin signal, similar to HTLV-1 p12. Despite that p8, RorfII and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal, only p8 is found in the nucleolus. p8 location in the nucleolus is linked to a bipartite NoLS. p8, and to a lesser extent p9, repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax3 and APH-3 promote cellular transformation. Altogether, these results complete the characterization of the newly described PTLV-3 virus.

    IMPORTANCE Together with their simian counterparts, HTLVs form the Primate T-Lymphotropic Viruses. HTLVs arose from interspecies transmission between non-human primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples or in vitro experiments, we have uncovered the presence of 3 unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four different proteins. We showed that two of them repress viral expression, but that none of them has the ability to induce colony formation. However, both Tax and the antisense encode protein APH-3 promote cell transformation. Our results allowed us to characterize for the first time 4 new retroviral proteins.

  • HIV-1 and morphine regulation of autophagy in microglia: Limited interactions in the context of HIV-1 infection and opioid abuse. [PublishAheadOfPrint]

  • Microglia are the predominant resident central nervous system (CNS) cell type productively infected by human immunodeficiency virus (HIV) type-1, and play a key role in the progression of HIV-associated dementia (HAD). Moreover, neural dysfunction and progression to HAD are accelerated in opiate drug abusers. In the present study, we examined the role of the autophagy pathway in the neuropathogenesis of HIV-1 using primary human microglial cells and determined whether opiates converge at this point. Infection of microglia with the HIV-1SF162 macrophage (M)-tropic strain resulted in increased Beclin1 expression, accompanied by an increase of LC3 protein levels and accumulation of LC3 reporter RFP+GFP+ (yellow) punctae, suggesting that HIV-1 infection triggers autophagosome formation without promoting protein degradation by the lysosome. Conversely, co-exposure with HIV-1 and morphine significantly decreased viral-induced Beclin1 expression and autophagosome formation. Exploration of the possible mechanism(s) used by morphine to disrupt the autophagic process unveiled a significant increase in intracellular pH, which coincided with a reduction in the formation of acidic vesicular organelles and in autophagolysosome formation. Small interfering (si) RNA targeting BECN1, a gene critical for autophagosome formation, significantly reduced viral replication and the viral-induced inflammatory responses. Conversely, morphine-enhanced viral replication and inflammatory responses were not affected by gene silencing with siBeclin1, suggesting that the interactive effect of morphine in HIV-1 pathogenesis is mediated through a Beclin1-independent mechanism. These novel findings may have important implications on the connections between autophagy and HIV-1 pathogenesis mediated by microglial cells in opioid-abusing individuals.

    IMPORTANCE About 50% of individuals infected with HIV-1 will develop some sort of neurocognitive impairment that cannot be prevented nor eradicated by antiretroviral therapy. The neuropathogenesis is mostly due to inflammatory responses by infected microglia, the resident immune cells of the brain. Cognitive disorders may also be associated with drugs of abuse. In fact, opioid drug users have an increased risk of developing neurocognitive disorders with increased progression to dementia. While the mechanism(s) by which opioids exacerbate the neuropathogenesis of HIV-1 are not entirely known, it is well accepted that glia are critical to opiate responses. This study gives us new insight into possible autophagic mechanism(s) in microglia that control HIV-1 replication and viral-induced inflammation in the context of opioid abuse and should greatly improve our knowledge in the pathogenesis of HIV-1 resulting from substance abuse to provide a better understanding for the design of candidate antiviral therapies targeting drug-abusing individuals.

  • Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of ILK and Cardiovascular Defects in Zebrafish [PublishAheadOfPrint]

  • Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality to mandarin fish (Siniperca chuatsi) in China and South-East Asia. Up to now, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three ankyrin-repeats (3ANK) domain-containing protein, which shows high similarity to the dominant-negative form of integrin-linked kinase (ILK), i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in Fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of sarcomeric Z-disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by over-expressing a dominant-negative form of ILK or by injecting an ILK antisense morpholino. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z-disk in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L overexpressing zebrafish embryos. As such, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis.

    IMPORTANCE: Our work is the first to show the role of a dominant-negative inhibitor of the host ILK from ISKNV (an Iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs the ILK signalling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z-disk in cadiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

  • Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head [PublishAheadOfPrint]

  • Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F-cavity recently proposed to contribute to the interaction with the H-protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H-binding and fusion-triggering deficiencies when mutated in combination. Since both residues reside on one side of the F-cavity, the data suggest that H binds the F-globular head domain sideway.

  • Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important co-factors that promote L protein stability and RNA synthesis. [PublishAheadOfPrint]

  • The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its co-factor the phosphoprotein (P) which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. Whilst cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, EGFP-tagged L and P protein expression was coupled with high affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L or the P proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein, including in both cases, protein chaperones. Ablation of chaperone activity using small molecule inhibitors confirmed previous studies, which suggested this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 was critical for L protein function and stability, whether in the presence or absence of the P protein. Inhibition studies suggested that HSP70 also disrupted virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a pro-viral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication.

    Importance Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription with a view to providing potential targets for the development of a specific, transient therapeutic, which disrupts virus biology but prevents the emergence of resistance whilst maintaining cell viability. In particular, protein chaperones (heat shock protein 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested and this study demonstrates to the field, that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.

  • PDZRN3/LNX3 is a Novel Target of HPV 16 and HPV-18 E6 [PublishAheadOfPrint]

  • High-risk HPV E6 proteins have a C-terminal PDZ binding motif, through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recently, studies suggested that the RING-containing ubiquitin-protein ligase, PDZRN3, might also be an HPV E6 target. In this analysis we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ and proteasome-dependent manner, and provides a connection between the HPV life-cycle and differentiation-related STAT-signalling.

  • Role of ATM in the Formation of Replication Compartment During Lytic Replication of EBV in Nasopharyngeal Epithelial Cells [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently-infected cells induces ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic genes transcription to promote lytic reactivation. Its contribution to the formation of replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using siRNA approach or specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virion in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at Serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of ATM-dependent DDR pathway in lytic reactivation of EBV suggesting a potential anti-viral replication strategy using specific DDR inhibitors.

    Importance Epstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC) which is of high prevalence in Southern China. EBV can either establish latent or lytic infection depends on the cellular context of infected host cells. Recent studies have highlighted the importance of DNA damage response (DDR), a surveillance mechanism that evolves to maintain genome integrity, in regulating lytic EBV replication. However, the underlying molecular events are largely undefined. The ATM is consistently activated in EBV-infected epithelial cells when induced to undergo lytic reactivation. Suppression of ATM inhibits replication of viral DNA. Furthermore, we observed that phosphorylation of Sp1 at Serine-101 residue, a downstream event of ATM activation, plays essential role in the formation of viral replication compartment for replication of virus DNA. Our study provides new insights into the mechanism of how EBV utilizes host cell machinery to promote replication of viral DNA upon lytic reactivation.

  • Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells [PublishAheadOfPrint]

  • After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the CsA washout assay, in which TRIM-CypA mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1 infected cells. Here we have extended this analysis to examine the effect of p24CA mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24CA mutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to a similar extent as wildtype with all the p24CA mutants tested. In addition, we observed differences in uncoating in two cell lines suggesting that cellular environment can differentially impact the disassembly of wildtype and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating.

    Importance The HIV-1 capsid is a conical shaped structure, composed of the HIV-1 encoded protein p24CA, which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study we used an in vivo assay to examine the uncoating process in HIV-1 infected cells. We determined that p24CA mutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24CA mutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

  • Coxsackievirus B3 Engineered to Contain microRNA Targets for Muscle-specific microRNAs Display Attenuated Cardiotropic Virulence in Mice [PublishAheadOfPrint]

  • Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5' untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development.

    Importance statement Coxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ~21 nucleotidemmdash;long, tissue-specific endogenous small RNA molecules that post-transcriptionally regulate gene expression by imperfectly binding to the 3' untranslated region (UTR), 5'-UTR, or coding region within a gene. In our study, muscle specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis, and may be useful for viral attenuation and vaccine development.

  • The HIV-1 gp120 CD4-bound conformation is preferentially targeted by ADCC-mediating antibodies in sera from HIV-1-infected individuals. [PublishAheadOfPrint]

  • Recent studies have linked antibody Fc-mediated effector functions with protection or control of HIV-1 and SIV infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in the Thai RV144 vaccine trial was suggested to correlate with a decreased HIV-1 acquisition risk. These antibodies were recently found to recognize HIV envelope (Env) epitopes exposed upon Env mmdash; CD4 interaction. CD4 downregulation by Nef and Vpu, as well as Vpu-mediated BST-2 antagonism, were reported to modulate exposure of those CD4-induced HIV-1 Env epitopes and therefore were proposed to play a role in reducing the susceptibility of infected cells to ADCC mediated by this class of antibodies. Here we report a high prevalence of antibodies recognizing CD4-induced HIV-1 Env epitopes in sera from HIV-1 infected individuals, which correlated with their ability to mediate ADCC responses against HIV-1 infected cells exposing these Env epitopes at the cell surface. Furthermore, our results indicate that Env variable regions V1, V2, V3 and V5 do not represent a major determinant for ADCC responses mediated by sera from HIV-1-infected individuals. Altogether, these findings suggest that HIV-1 tightly controls the exposure of certain Env epitopes at the surface of infected cells in order to prevent elimination by Fc-effector functions.

    Importance Here we identified a particular conformation of HIV-1 Env that is specifically targeted by ADCC-mediating antibodies present in sera from HIV-1-infected individuals. This observation suggests that HIV-1 developed sophisticated mechanisms to minimize the exposure of these epitopes at the surface of infected cells.

  • The sheep tetherin paralog, oBST2B, blocks envelope glycoprotein incorporation into nascent retroviral virions [PublishAheadOfPrint]

  • Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane similar to the llsquo;tetheringrrsquo; mechanism exerted by human BST2. In this study, we provide evidence that, unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A.

    Importance Bone marrow stromal cell antigen 2 (BST2) is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from BST2 proteins of other species.

  • Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus [PublishAheadOfPrint]

  • Until the recent emergence of two human pathogenic tick-borne phleboviruses (TBPVs) (Severe Fever with Thrombocytopenia Syndrome virus (SFTSV), and Heartland virus), TBPVs have been neglected as causative agents of human disease. In particular, no studies have addressed the global distribution of TBPVs and consequently, our understanding of the mechanism(s) underlying their evolution and emergence remains poor. In order to provide a useful tool for the ecological and epidemiological study of TBPVs, we have established a simple system that can detect all known TBPVs, based on conventional RT-PCR with degenerate primer sets targeting conserved regions of the viral L genome segment. Using this system, we have determined that several viruses that had been isolated from ticks decades ago but had not been taxonomically identified, are novel TBPVs. Full-genome sequencing of these viruses revealed a novel fourth TBPV cluster distinct from the three known TBPV clusters (i.e. the SFTS, Bhanja, and Uukuniemi groups) and from the mosquito/sandfly-borne phleboviruses. Furthermore, by using tick samples collected in Zambia, we confirmed that our system had enough sensitivity to detect a new TBPV in a single tick homogenate. This virus, tentatively designated as Shibuyunji virus after the region of tick collection, grouped into a novel fourth TBPV cluster. These results indicate that our system can be used as a first-line screening approach for TBPVs and that this kind of work will undoubtedly lead to the discovery of additional novel tick viruses and will expand our knowledge of the evolution and epidemiology of TBPVs.

    Importance Tick-borne phleboviruses (TBPVs) have been largely neglected until the recent emergence of two virulent viruses, Severe Fever with Thrombocytopenia Syndrome virus and Heartland virus. Little is known about the global distribution of TBPVs or how these viruses evolved and emerged. A major hurdle to study the distribution of TBPVs is the lack of tools to detect these genetically divergent phleboviruses. In order to address this issue, we have developed a simple, rapid, and cheap RT-PCR system that can detect all known TBPVs and which led to the identification of several novel phleboviruses from previously uncharacterized tick-associated virus isolates. Our system can detect virus in a single tick sample as well as novel TBPVs that are genetically distinct from any of the known TBPVs. These results indicate that our system described will be a useful tool for the surveillance of TBPVs and will facilitate understanding of the ecology of TBPVs.

  • Separate cellular localizations of HTLV-1 Env and GLUT1 are required for HTLV-1 Env-mediated fusion and infection [PublishAheadOfPrint]

  • Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection were also inhibited without overexpression of GLUT1, indicating that localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in separate intracellular compartments from Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition is probably due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env.

    Importance The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhance the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of the cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.

  • Persistent HCV infection impairs ribavirin antiviral activity through clathrin-mediated trafficking of equilibrative nucleoside transporter 1 [PublishAheadOfPrint]

  • Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit HCV replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently infected HCV cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda (IFN-1), a Type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, down-regulated membrane expression of ENT1 and terminated RBV uptake. In contrast, autophagy inhibitors hydroxychloroquine (HCQ), 3-Methyladenine (3-MA), and Bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces the ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently infected HCV cell cultures, and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection.

    IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism why RBV antiviral activity is impaired in persistently infected HCV cell culture, and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among Virologists, but also among the general public.

  • Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice, however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss, but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein.

    Importance Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line19 critical for fusion activity, and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.

  • Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex [PublishAheadOfPrint]

  • Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C to disrupt the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry we detected UL97-dependent phosphorylation of UL50 residue S216 and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions of both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells, and decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC, and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of nuclear lamina and phosphorylation of the NEC.

    Importance Human cytomegalovirus (HCMV) causes birth defects, and life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus, and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. Validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

  • Activation of ACLY by mTOR Signal Induces Disturbed Lipid Metabolism in HBV Pre-S2 Mutant Tumorigenesis [PublishAheadOfPrint]

  • The development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been found to be associated with disturbed lipid metabolism. To elucidate the role of lipid metabolism in HBV tumorigenesis, we investigated the dynamic pattern of lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis. Lipid and gene expression profiles were analyzed in an in vitro culture system and in transgenic mice livers harboring HBV pre-S2 mutant. The pre-S2 mutant transgenic livers showed a biphasic pattern of lipid accumulation, starting from mild fatty change in early (1 month) transgenic livers, which subsided and then remarkably increased in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACLY) activation. Further analyses revealed that the pre-S2 mutant initiated an endoplasmic reticulum (ER) stress-dependent mammalian target of rapamycin (mTOR) signal cascade. The pre-S2 mutant-induced mTOR signal activated the sterol regulatory element binding transcription factor 1 (SREBF1) to upregulate ACLY, which then activated the fatty acid desaturase 2 (FADS2) mediated through ACLY-dependent histone acetylation. Such an ER stress-dependent mTOR signal cascade is also important for the proliferation of hepatocytes in vitro and is further validated in HBV-related HCC tissues.

    Importance Aberrations of lipid metabolism frequently occur in chronic HBV infection. Our results provide the potential mechanism of disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis, which should be valuable for the design of HCC chemoprevention in high-risk HBV carriers.

  • Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes [PublishAheadOfPrint]

  • Autophagy is an ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit.

    Upon Human Immunodeficiency Virus-1 (HIV-1) infection, viral envelope glycoproteins (Env) induce autophagy-dependent apoptosis of uninfected bystander CD4+ T lymphocytes, a mechanism likely contributing to the loss of CD4+ T cells. In contrast, in productively infected CD4+ T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4+ T lymphocytes. Here we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat, but this process is rapidly counteracted by the virus to favor its replication and spread.

    IMPORTANCE Autophagy is recognized as one of the most ancient and conserved mechanism of cellular defense against invading pathogens. Crosstalks between HIV-1 and autophagy have been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4+ T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through an ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an anti-viral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

  • Expression of neuronal CXCL10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines/cytokines and enhancement of Blood-brain Barrier permeability [PublishAheadOfPrint]

  • It has been shown that enhancement of Blood-brain Barrier (BBB) permeability is modulated by expression of chemokines/cytokines and reduction of tight junction (TJ) proteins in the brains of mice infected with rabies virus (RABV). Since CXCL10 was found to be the most highly expressed chemokine, its temporal and spatial expression was determined in the present study. The expression of the chemokine CXCL10 was initially detected in neurons as early as 3 day post infection (dpi) before it was detected in microglia (6 dpi) and astrocytes (9 dpi) in the brain of RABV-infected mice. Neutralization of CXCL10 by treatment with anti-CXCL10 antibodies reduced IFN- production and Th17 cell infiltration as well as restored TJ protein expression and BBB permeability. Together, these data suggest that it is the neuronal CXCL-10 that initiates the cascade, leading to the activation of microglia/astrocytes, infiltration of inflammatory cells, expression of chemokines/cytokines, reduction of TJ protein expression, and enhancement of the BBB permeability.

  • Bindarit, an inhibitor of monocyte chemotactic proteins (MCPs) synthesis, protects against bone loss induced by Chikungunya virus infection [PublishAheadOfPrint]

  • The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used micro-computed tomographic (mmu;CT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in proximal tibial epiphysis of CHIKV-infected mice compared to mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-B ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. Expression levels of the monocyte chemoattractant proteins (MCPs) including, MCP-1/CCL2, MCP-2/CCL8 and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCPs inhibitor, bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphaviral-induced bone loss.

    IMPORTANCE Arthritogenic alphaviruses including chikungunya virus (CHIKV) and Ross River virus (RRV) cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host pro-inflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) are important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicate in bone and trigger bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints and bone loss can be ameliorated by treatment with an MCP drug inhibitor, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favouring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.

  • Clonal evolution of CD8+ T cell responses against latent viruses: relationship between phenotype, localization and function. [PublishAheadOfPrint]

  • Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, IL-7Raalpha;-expressing cells contain the precursors for long-lived antigen-experienced CD8+ T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here we studied whether IL-7Raalpha;-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool.

    Using flowcytometry and functional assays we found that the IL-7Raalpha;+ hCMV-specific T cell population comprises cells that have a memory-phenotype and lack effector-features. We used next-generation sequencing of the T-cell receptor to compare the clonal repertoires of IL-7Raalpha;+ and IL-7Raalpha;- subsets. We observed limited overlap of clones between these subsets during acute infection and after one year. When comparing the hCMV-specific repertoire between PB and paired LN we found many identical clones, but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only seldomly identical to the unique LN clones.

    Thus, although PB IL-7Raalpha;-expressing and LN hCMV-specific CD8+ T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8+ T-cell pool during latency or upon antigen recall. IL-7Raalpha;+ PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8+ effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naiiuml;ve CD8+ T cell pool.

    Importance Insight into the self-renewal properties of long-lived memory CD8+ T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is however not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: the circulating acute phase IL-7Raalpha;-expressing hCMV-specific CD8+ T cells and the lymph node (LN)-residing hCMV-specific (central-)memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation do only seldomly originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues, or recruited from the naiiuml;ve CD8+ T cell pool.

  • Virulence Determinants in the PB2 Gene 1 of a Mouse-Adapted H9N2 Virus [PublishAheadOfPrint]

  • The molecular basis of adaptation and pathogenicity of H9N2 influenza virus in mammals is largely unknown. Here we show that a mouse-adapted PB2 gene with a phenylalanine-to-leucine mutation (F404L) mainly contributes to the enhanced polymerase activity, replication, and pathogenicity of H9N2 in mice, and also increased the virulence of H5N1 and 2009 pandemic H1N1 influenza viruses. Therefore, we defined a novel pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.

  • A View of the E2--CD81 Interface at the Binding Site of a Neutralizing Antibody against Hepatitis C Virus [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses, in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W437LAGLF442, which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues, F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W437LAGLF442 helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for the antibody-mediated neutralization of HCV.

    Importance Characterization of the interface established between the virus and host cells can provide important information towards the control of virus infections. The interface formed that enables hepatitis C virus (HCV) to infect human liver cells has not been well-understood because of the number of cell surface proteins, factors and conditions found to be associated with the infection process. Based on a series of biochemical analyses, in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by the hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for the antibody-mediated neutralization of HCV.

  • Entry mechanisms of Herpes Simplex Virus Type 1 into murine epidermis: Involvement of nectin-1 and HVEM as cellular receptors [PublishAheadOfPrint]

  • Skin keratinocytes represent a primary entry site for herpes simplex virus type 1 (HSV-1) in vivo. The cellular proteins nectin-1 and HVEM act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin, and whether both nectin-1 and HVEM are involved, is not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1 deficient epidermis entry was strongly reduced. Almost no entry was observed, however, in nectin-1 deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1 deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis while HVEM has a more limited role. In primary murine keratinocytes where nectin-1 acts as single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we conclude that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, however, the restricted presence of HVEM can potentially replace nectin-1 as receptor illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo.

    Importance Herpes simplex virus (HSV) can cause a range of diseases in humans from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contribution of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1 or HVEM deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV type 1 (HSV-1) entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unravelling HSV invasion skin, which, in turn, allows the development of antiviral reagents.

  • Selective acquisition of host-derived ICAM-1 by HIV-1 is a matrix-dependent process [PublishAheadOfPrint]

  • HIV-1 acquires an impressive number of foreign components during its formation. Despite all the previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane protein found associated with mature virions, the Pr55Gag precursor polyprotein appears as a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55Gag-based virus-like particle (VLP) model. Substitution of various domains of Pr55Gag, such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural Matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two thirds of MA are important, and especially thirteen amino acids positioned inside the fifth aalpha;-helix. Moreover, based on 3D-modeling of protein-protein interactions (i.e. protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight on the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether these observations offer a new avenue for the development of anti-viral therapeutics that are directed at a target of host origin.

    Importance Intercellular adhesion molecule I (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase substantially HIV-1 infectivity. Despite all the previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remain(s) obscure. Previous data suggest that the Pr55Gag precursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed in whole virus preparations amplified in primary human cells, thereby providing physiological significance to our data.

  • Identification of TRIM27 as a Novel Degradation Target of Herpes Simplex Virus 1 ICP0 [PublishAheadOfPrint]

  • The herpes simplex virus 1 immediate-early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification as well as eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBS1 and MRE11, regulators of apoptosis including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal co-immunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions.

    Importance During productive infection a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To orchestrate such complex regulation, viruses including herpes simplex virus 1 (HSV-1) rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein regulates various cellular pathways concurrently by targeting a diverse set of cellular factors for degradation. While some of these targets have been previously identified and characterized, we undertook a proteomic screen to identify additional targets of this activity to further characterize ICP0's role during infection. We describe a set of candidate interacting proteins of ICP0 identified through this approach as well as our characterization of the most statistically significant result, the cellular transcriptional repressor TRIM27. We present TRIM27 as a novel degradation target of ICP0 and describe the relationship of these two proteins during infection.

  • Kaposi's Sarcoma-Associated Herpesvirus-Encoded RTA Impairs Innate Immunity via Ubiquitin-mediated Degradation of MyD88 [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a human -herpesvirus with latent and lytic reactivation cycles. The mechanism by which KSHV evades the innate immune system to establish latency has not yet been precisely elucidated. Toll-like receptors (TLRs) are the first line of defense against viral infections. Myeloid differentiation factor 88 (MyD88) is a key adaptor that interacts with all TLRs except TLR3 to produce inflammatory factors and type I interferons (IFNs), which are central components of innate immunity against microbial infection. Here, we found that KSHV replication and transcription activator (RTA), which is an immediate-early master switch protein of viral cycles, downregulates MyD88 expression at the protein level by degrading MyD88 through the ubiquitin (Ub)-proteasome pathway. We identified the interaction between RTA and MyD88 in vitro and in vivo and demonstrated that RTA functions as an E3 ligase to ubiquitinate MyD88. MyD88 was also repressed at the early stage of de novo infection as well as in lytic reactivation. We also found that RTA inhibited lipopolysaccharide (LPS)-triggered activation of the TLR4 pathway by reducing IFNs production and NF-B activity. Finally, we showed that MyD88 promoted the production of IFNs and inhibited KSHV LANA-1 gene transcription. Taken together, our results suggest that KSHV RTA facilitates the virus to evade innate immunity through degradation of MyD88, which might be critical for viral latency control.

    Importance: MyD88 is an adaptor for all TLRs other than TLR3 and mediates inflammatory factors and IFNs production. Our study demonstrated that the KSHV RTA protein functions as an E3 ligase to degrade MyD88 through the ubiquitin-proteasome pathway and block the transmission of TLRs signals. Moreover, we found that KSHV inhibited MyD88 expression during the early stage of de novo infection as well as in lytic reactivation. These results provide a potential mechanism for the virus to evade innate immunity.

  • Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming [PublishAheadOfPrint]

  • Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while non-enveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated as NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant-negative inhibition of the class XI myosins, XI-1, XI-2 and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localized in the cytoplasm but in close proximity to the ER membrane to form enveloped particles, and that this caused passive movements of cytoplasmic NBs by ER streaming.

    IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors, but have failed to provide evidence for the mechanism or biological significance of their motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

  • Grouper heat-shock cognate protein 70 (GHSC70) is involved in the cellular entry of nervous necrosis virus (NNV) [PublishAheadOfPrint]

  • Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish, and has affected more than 40 fish species. NNV belongs to the betanodavirus of Nodaviridae and is a non-enveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, the knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat-shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were presumably to be NNV receptor protein candidates. We cloned, sequenced, and expressed the genes of GHSC70 and GVDAC2 in Escherichia coli for anti-serum preparation. The expression knockdown of GHSC70 and GVDAC2 genes with specific short interfering RNA (siRNA) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. After an immuno-precipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 level after 1 h of adsorption to GF-1 cells, which was significantly lower in the cells pretreated with the GHSC70 antiserum than in non-treated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functions as NNV receptor or co-receptor protein.

    IMPORTANCE Fish nodavirus has caused mass mortality of more than 40 fish species worldwide, and resulted in huge economical lost in the past 20 years. Among the four genotypes of fish nodaviruses, red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we have developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating NNV-specific receptor(s) may exist on GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be NNV receptor (or co-receptor) candidate through VOBPA, and then provided several lines of evidence to demonstrate that GHSC70 protein was enrolled in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

  • Swine IFITM proteins potently inhibit influenza A virus replication [PublishAheadOfPrint]

  • Human IFITMs were identified as restriction factors of influenza A virus (IAV). Given the important role of pigs in the zoonotic cycle of IAV we cloned swine IFITMs (swIFITM) and found two IFITM1-like proteins, one homologue of IFITM2 and one of IFITM3. We show that swIFITM2 and swIFITM3 localize to endosomes and display potent antiviral activity. Knockdown of swIFITMs strongly reduced virus inhibition by interferon establishing the swIFITMs as potent restriction factors in porcine cells.

  • Phosphorylation of Herpes Simplex Virus 1 dUTPase Regulates Viral Virulence and Genome Integrity by Compensating for Low Cellular dUTPase Activity in the Central Nervous System [PublishAheadOfPrint]

  • A mutation in herpes simplex virus 1 dUTPase (vdUTPase), which precluded its phosphorylation at Ser-187, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low, and overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation. Thus, phosphorylation of vdUTPase appeared to regulate viral virulence and genome integrity by compensating for low cellular dUTPase activity in vivo.

    Importance Many DNA viruses encode a homolog of host cell dUTPases, which are known to function in accurate replication of cellular DNA genomes. The viral dUTPase activity has long been assumed to play a role in viral replication by preventing mutations in progeny virus genomes if cellular dUTPase activity was not sufficient. Here we showed that a mutation in herpes simplex virus 1 dUTPase, which precluded its phosphorylation at Ser-187 and reduced its activity, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low. In contrast, overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation in the brains of mice. This is the first report, to our knowledge, directly showing that viral dUTPase activity regulates viral genome integrity and pathogenicity by compensating for insufficient cellular dUTPase activity in vivo.

  • Profiling and characterization of potentially multidrug-resistant influenza neuraminidase 1 (N1) strains against neuraminidase inhibitors [PublishAheadOfPrint]

  • Neuraminidase inhibitors (NAIs) have been widely used to control influenza infection, but their increased use could promote global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened known mutation(s) that could confer multidrug resistance to currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA genes (catalytic residues, R152K and R292K; framework residues, E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single- and double-mutant viruses recovered in the backbone of pH1N1, 4 variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition to all the NAIs and 2 variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the 9 recombinant H5N1 viruses, 4 variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition to all the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance to pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.

    Importance There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition to all the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza variants as well as the development of alternative therapeutics.

  • Murine cytomegalovirus is not restricted to the house mouse Mus musculus domesticus: prevalence and genetic diversity in the European house mouse hybrid zone. [PublishAheadOfPrint]

  • The murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus (Mmd). It is a common infection in wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts and it is unknown if other Mus taxa carry the MCMV or if it is restricted to Mmd. We sampled mice over a 145km transect of Bavaria-Bohemia crossing an hybrid zone between Mmd and M. m. musculus (Mmm), to investigate the occurrence of MCMV in two Mus subspecies and test the limit of specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have co-diverged with their hosts and would have a geographical distribution constrained by the host genetic background. 520 mice were tested by ELISA and/or nested PCR targeting the M94 gene. Seropositive and PCR positive individuals were found in both Mus subspecies. Seroprevalence was high, 79.4%, but viral DNA was only detected in 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone showing one and two MCMV lineages in Mmd and Mmm, respectively. The estimated MCMV time to most recent common ancestor (1.1 Mya) matches that of their hosts. In conclusion, MCMV has co-evolved with these hosts suggesting its diversity in nature may be underappreciated as other members of the subgenus Mus likely carry different MCMVs.

    Importance The murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus, an important lab model for human cytomegalovirus (HCMV) infection. The majority of lab studies are based on only two strains of MCMVs isolated from M. m. domesticus, Smith and K181, itself derived from repeated passage of Smith in mouse submaxillary glands. The presence of MCMV in other members of the Mus subgenus had not even been investigated. By screening mouse samples collected in the European house mouse hybrid zone between M. m. domesticus and M. m. musculus, we show that MCMV is not restricted to the M. m. domesticus subspecies and that MCMV likely co-diverged with their Mus hosts. Thus the diversity of MCMV in nature may be seriously underappreciated since other members of the subgenus Mus likely carry their own MCMV lineages.

  • Slaying the Trojan horse: Natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T-cells [PublishAheadOfPrint]

  • Many attempts to design prophylactic HIV-1 vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus both in vitro and in vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of NK cells to respond in an Ab-dependent manner to allogeneic T-cells bearing HIV-1 antigens. This manuscript presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets, and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+ NK cell subset from HLA-Bw4+ individuals exhibits an activation advantage over the KIR3DL1nndash; subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses, and are potentially valuable for designing Ab-dependent therapies and/or vaccines.

    Importance: NK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression, however their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self inhibitory receptors. Here we show that with Ab-dependent stimulation NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

  • Contact-induced mitochondria polarization supports HIV-1 virological synapse formation [PublishAheadOfPrint]

  • Rapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles including mitochondria to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remains unclear. Here we report that contact between an HIV-1 infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live cell imaging we show that mitochondria and Gag polarization in HIV-1 infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts and is an active, calcium-dependent process. We also find that perturbation of mitochondria polarization impairs cell-cell spread of HIV-1 at the VS. Taken together these data suggest that HIV-1 infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.

    IMPORTANCE HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the Virological Synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles including mitochondria. Here we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondria-dependent increase in intracellular calcium, indicative of cellular signalling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

  • Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7 [PublishAheadOfPrint]

  • Susceptibility to alphavirus infection is age-dependent and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells that can be differentiated in vitro. Differentiation was associated with a 150- to 1000-fold decrease in replication of Sindbis and Venezuelan equine encephalitis alphaviruses, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein, but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor-3 and -7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant-negative isoform while that produced by differentiated neurons was the full-length aalpha; isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with up regulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication.

    IMPORTANCE Viral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old and New World alphaviruses and a bunyavirus were reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factors -3 and -7 mRNAs, and production of distinct isoforms of interferon regulatory factor-7 protein. Overall, our studies identify maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival.

  • Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals such as organ transplant recipients and infants infected in-utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a common target site on gB despite originating from different, healthy HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND-mutations in AD-5 were resistant to virus neutralizing MAbs. Competition ELISAs with human HCMV-convalescent sera from unselected donors confirmed the conserved antibody responses for the YNND epitope in HCMV infected individuals and, because a significant fraction of the gB-AD-5 response was directed against the YNND epitope, further argued that this epitope was a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed similar reactivity to AD-5 and comparable neutralization activity towards gB-mutant viruses as AD-5 specific MAbs. Taken together, our data indicated that the YNND epitope represents an important target for anti-gB antibody responses as well for anti-AD-5 virus neutralizing antibodies.

    Importance HCMV is a major global health concern and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies, e.g. vaccination. Mapping the target structures of neutralizing antibodies induced by naturally-occurring HCMV infection can aid in defining the properties required for a protective capacity of vaccine antigens. The data presented here extend our knowledge of neutralizing epitopes within gB to include AD-5. Collectively, our data will contribute to optimal vaccine design and development of antibody-based therapies.

  • Assessment of the internal genes of influenza A (H7N9) virus contributing to the high pathogenicity in mice [PublishAheadOfPrint]

  • The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants were generated by reverse genetics, each containing a single internal gene of the typical A/Anhui/1/2013(H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007(H9N2) virus. Their replication ability, polymerase activity, and pathogenicity were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed PB2-K627 is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed a greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface HA and NA proteins, contributing to the high infectivity of the H7N9 virus in humans.

    Importance To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

  • Topical HSV-2 Vaccination with HPV Vectors Expressing gB/gD Ectodomains Induces Genital Tissue Resident Memory CD8+ T cells and Reduces Genital Disease and Viral Shedding After HSV-2 Challenge [PublishAheadOfPrint]

  • No Herpes simplex virus type-2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular injection of HSV-2 gB and gD proteins in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) and gD (gDsec), but not PsV expressing cytoplasmic or membrane-bound forms, induced circulating and intravaginal tissue resident memory CD8+ T cells able to secrete IFN- and TNF-aalpha; and moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec/gDsec vaccines conferred improved survival after HSV-2 vaginal challenge compared to HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with reduced severity of genital lesions and lower viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with soluble HSV-2 gD in alum and monophosphoryl lipid A (gD2t/Alum/MPL) elicited high neutralizing antibody titers and improved survival, but did not reduce genital lesions and viral shedding. Vaccination combining HPV-gBsec/gDsec ivag and gD2t/Alum/MPL i.m. improved survival and reduced genital lesions and viral shedding. Finally, circulating HSV-2-specific CD8+ T cells, not serum antibodies, correlated with reduced viral shedding. Together our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.

    Importance: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a non-replicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV based vectors as a platform to induce genital tissue resident memory T cell responses and to control local manifestations of primary HSV infection.

  • Super-infection with woodchuck hepatitis virus (WHV) strain WHVNY of the livers chronically infected with the strain WHV7. [PublishAheadOfPrint]

  • The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all); virus super-infection is an unlikely event; and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Super-infection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced HCCs, were super-infected with another WHV strain, WHVNY. Six weeks after the super-infection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY super-infection was demonstrated by using WHV strain-specific PCR assays, and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all super-infected animals during weeks one through six after the super-infection; (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three super-infected woodchucks; and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the super-infection. The results are consistent with the occurrence of continuous, but inefficient hepadnavirus cell-to-cell spread and super-infection during chronic infection, and suggest that replication space occupied by the super-infecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection.

    IMPORTANCE. The study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus super-infection and cell-to-cell spread likely continue to occur; and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread are most likely mediated at the level of the cells, and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous, but limited hepadnavirus spread and super-infection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

  • Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors [PublishAheadOfPrint]

  • Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. MWPyV ST binds PP2A and LT binds pRb, p107, p130 and p53. However, MWPyV LT was less stable than SV40 LT and unable to promote the growth of normal cells. This study confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

  • Merkel cell polyomavirus small T antigen mediates microtubule destabilisation to promote cell motility and migration [PublishAheadOfPrint]

  • Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV Small and Large Tumour (ST and LT) antigens. Although a number of molecular mechanisms have been attributed to MCPyV tumour antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organisation and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilisation leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilisation and cell motility and implicate the cellular phosphatase catalytic subunit PP4C in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.

    IMPORTANCE Merkel Cell Polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally-induced cancer development are yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV Small Tumour (ST) antigen promotes the destabilisation of the host cell microtubule network which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin, by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel anti-tumoral therapies.

  • An interleukin-1 beta encoding retrovirus exhibits enhanced replication in vivo. [PublishAheadOfPrint]

  • Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1bbeta; secretion through the DNA-sensing molecule IFI16, the effect of IL-1bbeta; on the course of infection is unknown. To test whether IL-1bbeta; secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL1bbeta;) that encodes the mature form of IL-1bbeta;. This virus replicated with kinetics similar to wild-type virus in tissue culture, but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7dpi, mice infected with FMLV-IL1bbeta; exhibited splenomegaly and viral loads three hundred-fold higher than mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL1bbeta;-infected mice correlated with a large expansion of Gr-1+CD11b+ myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL1bbeta; infection was controlled by C57BL/6 mice by 14dpi, FMLV-IL1bbeta; was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1bbeta; secretion is a powerful positive regulator of retroviral infection, and that FMLV-IL1bbeta; represents a new model of pro-inflammatory retroviral infection.

    Importance Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1bbeta; on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL1bbeta;) that encodes IL-1bbeta; and promotes abundant IL-1bbeta; secretion from infected cells. This virus replicates with normal kinetics in cultured cells, but displays a dramatically enhanced ability to replicate in mice, and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1bbeta; as a positive regulator of retroviral replication and pathogenesis, and suggest that targeting this pathway may have therapeutic benefits for pro-inflammatory retroviruses. This virus can also be used to further study the impact of pro-inflammatory pathways on retroviral infection.

  • RHABDOVIRAL-BASED VACCINE PLATFORMS AGAINST HENIPAVIRUSES [PublishAheadOfPrint]

  • The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipaviridae family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms: recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type HeV glycoprotein (G). The RABV vector expressing the codon-optimized HeV G showed a 2 to 3-fold increase in incorporation compared to the RABV vector expressing wild-type (wt) HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vector. To test immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10mmu;g chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.

    Importance Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV) expressing wild-type or codon-optimized HeV glycoprotein, with the goal to develop a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.

  • G2/M cell cycle arrest correlates with primate lentiviral Vpr interaction with the SLX4 complex [PublishAheadOfPrint]

  • The accessory gene vpr, common to all primate lentiviruses, induces a potent G2/M arrest in cycling cells. A recent study showed that HIV-1 Vpr mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi Anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of SIV Vpr proteins. We show that SIV Vpr proteins vary in their ability to promote cell-cycle arrest in human cells. Whilst this is dependent on the DCAF1/DDB1/CUL4 Ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with huSLX4 and could be abolished by siRNA depletion of any member of the SLX4 complex. By contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in Grivet cells. Correspondingly, here cell-cycle arrest correlated with interaction with the Grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins, and that the ability to dysregulate the Fanconi Anemia DNA repair pathway is an essential function of Vpr in vivo.

    Importance The Vpr protein of HIV-1 and its related viruses is essential for the virus in vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data has shown that HIV-1 Vpr targets the Fanconi Anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA crosslinks. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell-cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. These studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins, and that the ability to dysregulate members of the Fanconi Anemia DNA repair pathway is essential for HIV/SIV replication in vivo.

  • Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype [PublishAheadOfPrint]

  • Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for development of new rationally designed alphavirus vaccine candidates, which combine efficient immunogenicity, high safety and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study, combine a variety of characteristics which independently contribute to a reduction of virulence. These constructs encode a noncytopathic VEEV capsid protein, which is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications additionally affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses.

    SIGNIFICANCE Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging and interaction with the host to design new VEEV vaccine candidates, which demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis, but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.

  • Tetramer-enrichment reveals presence of phenotypically diverse hepatitis C virus specific CD8+ T cells in chronic infection [PublishAheadOfPrint]

  • Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically genotype 1a infected, HLA-A*02:01+ patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector-memory phenotype whereas, surprisingly, one third displayed a naiiuml;ve-like phenotype despite ongoing viral replication. CD8+ T cells with an effector-memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naiiuml;ve-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naiiuml;ve-like CD8+ T cells from patients with consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence.

    Importance In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01 restricted epitopes in chronically HCV infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape and functional impairment of naiiuml;ve-like HCV-specific CD8+ T cells.

  • Quantitative proteomics identifies host factors modulated during acute hepatitis E infection in swine model [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes acute enterically-transmitted hepatitis. In industrialized countries, it is a zoonotic disease, swine being the major reservoir of human HEV contaminations. The occurrence and severity of the disease are variable, ranging from asymptomatic to self-limiting acute hepatitis, chronic infection or fulminant hepatitis. In the absence of a robust cell culture system or small animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with 3 different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, 2D-DIGE and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the 3 HEV strains replicate similarly in swine and that they modulate several cellular pathways suggesting that HEV impairs several cellular processes, which can account for various disease expressions. Several proteins such as Heterogeneous nuclear ribonucleoprotein K, Apolipoprotein E and Prohibitin, known to be involved in other viral life cycles, were up-regulated in HEV-infected livers. Some differences were observed between the 3 strains, suggesting that HEV's genetic variability may induce variation in pathogenesis. This comparative analysis of liver proteome modulated during HEV infection with 3 different strains of genotype 3 provides an important basis for further investigations on factors involved in HEV replication and the mechanism of HEV pathogenesis.

    Importance Hepatitis E virus (HEV) is responsible for acute hepatitismmdash;ranging from asymptomatic to self-limiting acute hepatitismmdash;chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, swine being the principal reservoir for human contaminations. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol, lipid metabolism or cell survival were modulated during HEV infection in swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.

  • A 2.5 kb deletion contaning a cluster of nine microRNAs in the LAT locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency. [PublishAheadOfPrint]

  • The alphaherpesvirus Pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within LAT, suggesting a role in establishment and/or maintenance of latency.

    We generated a mutant PrV (M) deleted of nine miRNA genes which displayed almost identical properties with the parental PrV (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT, M or mock infected.

    Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days post infection trigeminal ganglia were excised and profiled by deep sequencing and RT-qPCR.

    Latency was established in all infected animals without evidence of viral reactivation demonstrating that miRNAs are not mandatory for this process. Lower levels of Large Latency Transcript (LLT) were found in ganglia infected by M compared to WT PrV. All PrV miRNAs were expressed, with highest expression found for prv-miR-LLT1, prv-miR-LLT2 (in WT-ganglia) and prv-miR-LLT10 (in both WT and M-ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M and control ganglia. Both viruses triggered a strong host immune response, but in M- ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and migration of dendritic cells, were impaired in M- ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state.

    Importance This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV the only remarkable differences are found in the pattern of host response. This indicates that, LAT functions as an immune evasion locus.

  • Basic motifs target PSGL-1, CD43, and CD44 to plasma membrane sites where HIV-1 assembles [PublishAheadOfPrint]

  • HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in non-polarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color super-resolution localization microscopy. Consistent with the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with that of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants.

    Importance Nascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using super-resolution localization microscopy suggest the presence of a novel molecular mechanism promoting association of PSGL-1, CD43, and CD44 with assembling HIV-1, which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein.

  • Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells [PublishAheadOfPrint]

  • Influenza A viruses (IAVs) rely on host factors to support their life cycle as viral proteins could "hijack" or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase the knowledge of molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of influenza NS2 protein, the nuclear export protein responsible for overcoming host-range restriction, by a yeast two-hybrid screening, GST pull-down and co-immunoprecipitation assays, and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator for IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced stability of the matrix protein M1. We further provided evidence that AIMP2 recruitment switched the modification of M1 from ubiquitination to SUMOylation occurring on the same attachment site K242 on M1, and thereby promoted M1-mediated vRNPs nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 in mediating influenza virus replication.

    IMPORTANCE Although ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine K242 on M1 and interaction of NS2 with AIMP2 facilitates the switch of M1 modification from ubiquitination to SUMOylation thus increasing viral replication.

  • The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes [PublishAheadOfPrint]

  • The incidence of infection with any of the four dengue virus serotypes (DENV 1-4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an HLA-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. PBMCs from these donors were screened in IFN ELISPOT assays with pools of predicted, HLA matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, while broad responses to structural and non-structural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused towards the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.

    Importance The development of effective vaccination strategies against DENV infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that sero-conversion is the only reliable correlate of protection. Here, we show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

  • A novel mode of ISG15 mediated protection against influenza A virus and Sendai virus in mice [PublishAheadOfPrint]

  • ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15nndash;/nndash; mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15nndash;/nndash; mice. We found that ISG15 protects mice from virus induced lethality by a conjugation dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication, and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but non-antiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo.

    Importance It has previously been demonstrated that ISG15nndash;/nndash; mice are more susceptible to a number of viral infections. As one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. While a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15nndash;/nndash; mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo.

  • Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation [PublishAheadOfPrint]

  • The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intra-molecular interactions remains elusive. By solving the crystal structure of Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal 261-303 residues deleted. Comparing to the wild type (WT) NS5, full-length NS5 variants exhibited consistent up- or down-regulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266-275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276-303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface was relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.

    IMPORTANCE The flavivirus NS5 is very unique in placing a methyltransferase (MTase) to the immediate N-terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5 that reveals a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitioning. By comparing the N-terminal truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally un-clarified previously. Taken together, we have provided biochemical evidences and mechanistic insights for the crosstalk between the two enzyme modules of flavivirus NS5.

  • Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus Type 1 Myocarditis [PublishAheadOfPrint]

  • Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species-specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus type 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon-gamma (IFN-) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was predominantly composed of CD3+ T lymphocytes. Depletion of IFN- during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN- is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction.

    Importance Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN- modulation, as a therapeutic strategy.

  • Insight into the Mechanism of Inhibition of Recombinant Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex [PublishAheadOfPrint]

  • Adeno-Associated virus (AAV) is a dependent virus of the family parvoviridae. Gene expression and replication of AAV and derived recombinant vectors (rAAV) are severely limited (ggt;10-fold) by the cellular DNA damage sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). AAV does not encode the means to circumvent this block to productive infection, but relies on co-infecting helper-virus to do so. Using adenovirus helper proteins E1B55k andE4orf6, which enhance transduction of AAV via degradation of MRN, we investigate the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We test substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA-binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.

    IMPORTANCE Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell, but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on co-infecting helper-virus, such as Adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications toward understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance, and likely translate to other viruses that must navigate the DDR.

  • Activation of p90 Ribosomal S6 Kinases (RSKs) by ORF45 of Kaposi Sarcoma-Associated Herpesvirus is Critical for Optimal Production of Infectious Viruses [PublishAheadOfPrint]

  • We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and ERK. We have now identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK, and consequently its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant BAC16-45A66F. Reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion was also accompanied by noticeably decreased viral gene expression during lytic reactivation. The ORF45-F66A mutant consequently produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.

    IMPORTANCE STATEMENT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment, and facilitate efficient production of progeny viral particles. We have previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

  • Variation of pH sensitivity, acid stability, and fusogenicity of three influenza H3 subtypes [PublishAheadOfPrint]

  • Influenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein, hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single particle tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provides detailed information regarding HA pH sensitivity, acid stability, and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn) and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while fusion of the egg-adapted strains, Udorn and X:31, are strongly dependent on pH (and faster) as pH decreases. All strains exhibit similar acid stability (the length of time they remain fusogenic in an acidic environment) at higher pH's, but the egg-adapted strains become less acid stable at lower pH's. Thus, it appears that the lab-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain.

    Importance The ability of influenza virus to release its genome under different acidic conditions has recently been linked to transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are an intra-H3 subtype study of acid stability and fusion kinetics. Using a single particle tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH where the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time virions can persist in acidic environment and still be fusion competent.

  • The putative HSV-1 chaperone protein UL32 modulates disulfide bond formation during infection. [PublishAheadOfPrint]

  • During DNA encapsidation, HSV-1 procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28 and UL33) and one viral protein, UL32, not found associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional HA-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 nndash; 9 h post infection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell while at 23 hpi it was additionally found in the cytoplasm. A newly generated UL32 null mutant was used to confirm that although B-capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

    Importance Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32 null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.

  • Complex TCR repertoire dynamics underlie the CD8+ T-cell response to HIV-1 [PublishAheadOfPrint]

  • Although CD8+ T-cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naiiuml;ve individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, evidenced by discordant clonotype-specific evolution against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR/antigen interface.

    Importance We describe a relation between viral epitope mutation, antigen-specific T-cell expansion and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provide insights into the process of co-adaptation between the human immune system and a rapidly evolving lentivirus.

  • The Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for BV production and ODV envelopment [PublishAheadOfPrint]

  • ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11-knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5'-RACE analysis revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in the Ac11KO-transfected Sf9 cells. However, qPCR analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of the Ac11KO-transfected cells, which demonstrates that the defect in BV production from the Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, the electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form ODVs, and that their subsequent embedding into OBs was also blocked in the Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.

    IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vector. Although the function of baculovirus genes in viral replication has been studied using gene knock-out technology, the function of more than one-third of viral genes which include some highly conserved genes are still unknown. In this study, ac11 was proved to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

  • NS5A and human RPA increase the processivity of HCV NS5B polymerase activity in vitro [PublishAheadOfPrint]

  • The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling circle RNA template. Under close-to-physiological salt conditions, HCV NS5B21, the RNA dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A, recruit NS5B21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5B21 resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5B21bbull;NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.

    Importance The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood but viral and host proteins collaborate in this process. Using a rolling circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5B21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors

  • Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-dependent RNA Polymerases [PublishAheadOfPrint]

  • Viral RNA-dependent RNA polymerases are considered to be low fidelity enzymes, providing for high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered over a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity, and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination and these are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the fingers domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower fidelity enzyme where analogous mutations now increase nucleotide discrimination. The findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that (+) strand RNA viruses retain a unique palm domain based active site closure mechanism to fine tune replication fidelity.

    IMPORTANCE Positive strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow for the rapid adaptation of these viruses to different tissue types and host cells. In this work we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. The results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. The findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

  • Expression of IFN{gamma} by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon [PublishAheadOfPrint]

  • Previous animal model experiments have shown a correlation between interferon gamma (IFN) expression and both survival from infection with attenuated rabies virus and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBN. Morbidity and mortality were monitored in mice infected intranasally with SPBN or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN. Incorporation of IFN into the rabies genome highly attenuated the virus. SPBN has an LD50 more than 100 fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBN infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNARnndash;/nndash;) cannot control the SPBN infection and rapidly die. These data suggest that IFN production has antiviral effects in rabies, largely due to the induction of type I interferons.

    IMPORTANCE Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the CNS this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover antibody deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN directly to the infected tissue reduces rabies virus replication and spread limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

  • Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor [PublishAheadOfPrint]

  • The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74, and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.

    IMPORTANCE The HIV-1 capsid is an emerging drug target, and several small molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

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