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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]

  • Intercellular Transmission of Viral Populations with Vesicles [Gem]

  • A common paradigm holds that during cell-to-cell transmission, viruses behave as lone soldiers. Recently, we discovered not only that enteroviruses are transmitted via vesicles as populations of viral particles but also that this type of transmission enhances their infection efficiency (Y. H. Chen et al., Cell 160:619nndash;630, 2015). This mechanism could be advantageous for the overall fitness of the viral population, promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here, we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is widespread among different virus families and includes populations of both viral particles and naked viral genomes.

  • Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region [Structure and Assembly]

  • Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion.

    IMPORTANCE This study reveals conformational and antigenic differences between the 123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

  • Interplay between the Herpes Simplex Virus 1 gB Cytodomain and the gH Cytotail during Cell-Cell Fusion [Virus-Cell Interactions]

  • Herpesvirus entry into cells is mediated by the viral fusogen gB, which is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. In contrast to most viral fusogens, gB requires a conserved heterodimer, gH/gL, as well as other nonconserved proteins. In a further mechanistic twist, gB-mediated cell-cell fusion appears restricted by its intraviral or cytoplasmic domain (cytodomain) because mutations within it result in a hyperfusogenic phenotype. Here, we characterized a panel of hyperfusogenic HSV-1 gB cytodomain mutants and show that they are fully functional in cell-cell fusion at shorter coincubation times and at lower temperatures than those for wild-type (WT) gB, which suggests that these mutations reduce the kinetic energy barrier to fusion. Despite this, the mutants require both gH/gL and gD. We confirm previous observations that the gH cytotail is an essential component of the cell-cell fusion mechanism and show that the N-terminal portion of the gH cytotail is critical for this process. Moreover, the fusion levels achieved by all gB constructs, WT and mutant, were proportionate to the length of the gH cytotail. Putting these results together, we propose that the gH cytotail, in addition to the gH/gL ectodomain, plays an essential role in gB activation, potentially acting as a "wedge" to release the gB cytodomain "clamp" and enable gB activation.

    IMPORTANCE Herpesviruses infect their hosts for life and cause a substantial disease burden. Herpes simplex viruses cause oral and genital sores as well as rare yet severe encephalitis and a panoply of ocular ailments. Infection initiates when the viral envelope fuses with the host cell membrane in a process orchestrated by the viral fusogen gB, assisted by the viral glycoproteins gH, gL, and gD and a cellular gD receptor. This process is more complicated than that of most other viruses and is subject to multiple regulatory inputs. Antiviral and vaccine development would benefit from a detailed mechanistic knowledge of this process and how it is regulated.

  • Genetic Diversity within Alphaherpesviruses: Characterization of a Novel Variant of Herpes Simplex Virus 2 [Genetic Diversity and Evolution]

  • Very low levels of variability have been reported for the herpes simplex virus 2 (HSV-2) genome. We recently described a new genetic variant of HSV-2 (HSV-2v) characterized by a much higher degree of variability for the UL30 gene (DNA polymerase) than observed for the HG52 reference strain. Retrospective screening of 505 clinical isolates of HSV-2 by a specific real-time PCR assay targeting the UL30 gene led to the identification of 13 additional HSV-2v isolates, resulting in an overall prevalence of 2.8%. Phylogenetic analyses on the basis of microsatellite markers and gene sequences showed clear differences between HSV-2v and classical HSV-2. Thirteen of the 14 patients infected with HSV-2v originated from West or Central Africa, and 9 of these patients were coinfected with HIV. These results raise questions about the origin of this new virus. Preliminary results suggest that HSV-2v may have acquired genomic segments from chimpanzee alphaherpesvirus (ChHV) by recombination.

    IMPORTANCE This article deals with the highly topical question of the origin of this new HSV-2 variant identified in patients with HIV coinfection originating mostly from West or Central Africa. HSV-2v clearly differed from classical HSV-2 isolates in phylogenetic analyses and may be linked to simian ChHV. This new HSV-2 variant highlights the possible occurrence of recombination between human and simian herpesviruses under natural conditions, potentially presenting greater challenges for the future.

  • Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-{kappa}B Response Elements [Genome Replication and Regulation of Viral Gene Expression]

  • Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-B binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation.

    IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that costimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-B family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-B binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to repower the cycle of cytomegalovirus propagation.

  • Whole-Genome Sequencing of Kaposi's Sarcoma-Associated Herpesvirus from Zambian Kaposi's Sarcoma Biopsy Specimens Reveals Unique Viral Diversity [Genetic Diversity and Evolution]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countriesmmdash;where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization.

    IMPORTANCE Our results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

  • Induction and Antagonism of Antiviral Responses in Respiratory Syncytial Virus-Infected Pediatric Airway Epithelium [Cellular Response to Infection]

  • Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well-differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda 1 (IFN-1)/interleukin-29 (IL-29). In contrast, type I IFNs were not detected following RSV infection of WD-PBECs. IFN responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV nonstructural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic.

    IMPORTANCE Most respiratory viruses target airway epithelium for infection and replication, which is central to causing disease. However, for most human viruses we have a poor understanding of their interactions with human airway epithelium. Respiratory syncytial virus (RSV) is the most important viral pathogen of young infants. To help understand RSV interactions with pediatric airway epithelium, we previously developed three-dimensional primary cell cultures from infant bronchial epithelium that reproduce several hallmarks of RSV infection in infants, indicating that they represent authentic surrogates of RSV infection in infants. We found that RSV induced a potent antiviral state in these cultures and that a type III interferon, interleukin IL-29 (IL-29), was involved. Indeed, our data suggest that IL-29 has potential to prevent RSV disease. However, we also demonstrated that RSV efficiently circumvents this antiviral immune response and identified mechanisms by which this may occur. Our study provides new insights into RSV interaction with pediatric airway epithelium.

  • Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle [Virus-Cell Interactions]

  • Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (M) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of M is impaired or the extent to which seasonal IAV replicate in M. Herein, we compared mouse M and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse M. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected M, defining a second level of restriction late in the virus life cycle.

    IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages.

  • MDA5 Is Critical to Host Defense during Infection with Murine Coronavirus [Pathogenesis and Immunity]

  • Infection with the murine coronavirus mouse hepatitis virus (MHV) activates the pattern recognition receptors melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 7 (TLR7) to induce transcription of type I interferon. Type I interferon is crucial for control of viral replication and spread in the natural host, but the specific contributions of MDA5 signaling to this pathway as well as to pathogenesis and subsequent immune responses are largely unknown. In this study, we use MHV infection of the liver as a model to demonstrate that MDA5 signaling is critically important for controlling MHV-induced pathology and regulation of the immune response. Mice deficient in MDA5 expression (MDA5nndash;/nndash; mice) experienced more severe disease following MHV infection, with reduced survival, increased spread of virus to additional sites of infection, and more extensive liver damage than did wild-type mice. Although type I interferon transcription decreased in MDA5nndash;/nndash; mice, the interferon-stimulated gene response remained intact. Cytokine production by innate and adaptive immune cells was largely intact in MDA5nndash;/nndash; mice, but perforin induction by natural killer cells and levels of interferon gamma, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-aalpha;) in serum were elevated. These data suggest that MDA5 signaling reduces the severity of MHV-induced disease, at least in part by reducing the intensity of the proinflammatory cytokine response.

    IMPORTANCE Multicellular organisms employ a wide range of sensors to detect viruses and other pathogens. One such sensor, MDA5, detects viral RNA and triggers induction of type I interferons, chemical messengers that induce inflammation and help regulate the immune responses. In this study, we sought to determine the role of MDA5 during infection with mouse hepatitis virus, a murine coronavirus used to model viral hepatitis as well as other human diseases. We found that mice lacking the MDA5 sensor were more susceptible to infection than were mice with MDA5 and experienced decreased survival. Viral replication in the liver was similar in mice with and without MDA5, but liver damage was increased in MDA5nndash;/nndash; mice, suggesting that the immune response is causing the damage. Production of several proinflammatory cytokines was elevated in MDA5nndash;/nndash; mice, suggesting that MDA5 may be responsible for keeping pathological inflammatory responses in check.

  • Contribution of Epidemiological Predictors in Unraveling the Phylogeographic History of HIV-1 Subtype C in Brazil [Genetic Diversity and Evolution]

  • The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states, and the fact that HIV-1C infections increase at a higher rate than subtype B infections in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence data set sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil, considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled data sets in order to mitigate potential sample biases. We reveal a central role for the city of Porto Alegre, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northward expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burdens and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this.

    IMPORTANCE Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the south, where it now circulates as the dominant variant. The northward spread has been comparatively slow, and HIV-1B still prevails in that region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination has not yet been explored. Here, we complement publically available sequences and new genetic data from 13 cities with epidemiological data to reconstruct the history of HIV-1C spread in Brazil. The combined approach results in more robust reconstructions and can protect against sampling bias. We found evidence for an alternative view of the HIV-1C spatiotemporal history in Brazil that, contrary to previous explanations, integrates seamlessly with other observational data.

  • Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling [Virus-Cell Interactions]

  • Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection.

    IMPORTANCE RNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses.

  • Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains [Prions]

  • Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrPC). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrPC modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95+) with distinct biological properties. Transmission of first-passage tg60CWD-H95+ isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrPC primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrPC molecules results in the generation of novel CWD strains.

    IMPORTANCE CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrPC) to the emergence of a novel CWD strain (H95+). We show that, upon infection, deer expressing H95-PrPC molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95+ CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD.

  • The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain [Virus-Cell Interactions]

  • Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain.

    IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

  • Breakthrough Virus Neutralization Resistance as a Correlate of Protection in a Nonhuman Primate Heterologous Simian Immunodeficiency Virus Vaccine Challenge Study [Vaccines and Antiviral Agents]

  • Comprehensive assessments of immune correlates of protection in human immunodeficiency virus (HIV) vaccine trials are essential to vaccine design. Neutralization sieve analysis compares the neutralization sensitivity of the breakthrough transmitted/founder (TF) viruses from vaccinated and control animals to infer the molecular mechanisms of vaccine protection. Here, we report a robust neutralization sieve effect in a nonhuman primate simian immunodeficiency virus (SIV) vaccine trial (DNA prime/recombinant adenovirus type 5 [rAd5] boost) (VRC-10-332) that demonstrated substantial protective efficacy and revealed a genetic signature of neutralization resistance in the C1 region of env. We found significant enrichment for neutralization resistance in the vaccine compared to control breakthrough TF viruses when tested with plasma from vaccinated study animals, plasma from chronically SIV-infected animals, and a panel of SIV-specific monoclonal antibodies targeting six discrete Env epitopes (P llt; 0.008 for all comparisons). Neutralization resistance was significantly associated with the previously identified genetic signature of resistance (P llt; 0.0001), and together, the results identify virus neutralization as a correlate of protection. These findings further demonstrate the in vivo relevance of our previous in vitro analyses of the SIVsmE660 challenge stock, which revealed a broad range of neutralization sensitivities of its component viruses. In sum, this report demonstrates proof-of-concept that phenotypic sieve analyses can elucidate mechanistic correlates of immune protection following vaccination and raises a cautionary note for SIV and SHIV (simian-human immunodeficiency virus) vaccine studies that employ challenge strains with envelope glycoproteins that fail to exhibit neutralization resistance profiles typical of TF viruses.

    IMPORTANCE With more than 2 million new infections annually, the development of an effective vaccine against HIV-1 is a global health priority. Understanding immunologic correlates of protection generated in vaccine trials is critical to advance vaccine development. Here, we assessed the role of vaccine-elicited neutralizing antibodies in a recent nonhuman primate study of a vaccine that showed significant protection against simian immunodeficiency virus (SIV) challenge and suggested a genetic signature of neutralization sensitivity. We found that breakthrough viruses able to establish infection in vaccinated animals were substantially more resistant to antibody-mediated neutralization than were viruses from controls. These findings suggest that vaccine-elicited neutralizing antibodies selectively blocked the transmission of more sensitive challenge viruses. Sieve analysis also corroborated a genetic signature of neutralization sensitivity and highlighted the impact of challenge swarm diversity. Our findings suggest an important role for neutralization sieve analyses as an informative component of comprehensive immune-correlates analyses.

  • Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells [Virus-Cell Interactions]

  • Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development.

    IMPORTANCE A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.

  • Prion Infectivity Plateaus and Conversion to Symptomatic Disease Originate from Falling Precursor Levels and Increased Levels of Oligomeric PrPSc Species [Prions]

  • In lethal prion neurodegenerative diseases, misfolded prion proteins (PrPSc) replicate by redirecting the folding of the cellular prion glycoprotein (PrPC). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in presymptomatic disease is preceded by a progressive fall in PrPC level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrPSc). Although a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrPC levels and continuously increasing levels of oPrPSc are crucial determinants in the transition from presymptomatic to symptomatic prion disease.

    IMPORTANCE Prions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell surface protein, PrPC to a form called PrPSc. Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrPL or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrPC precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrPC, which transduces a toxic effect, along with the amount of oligomeric forms of PrPSc.

  • A Unique 5' Translation Element Discovered in Triticum Mosaic Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Several plant viruses encode elements at the 5' end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5' m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5' leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5' leader to a 300-nucleotide sequence that drives cap-independent translation from the 5' end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5' untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5' UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5' end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation.

    IMPORTANCE Many members of the Potyviridae family rely on their 5' end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5' leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5' UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5' UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

  • Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles [Virus-Cell Interactions]

  • Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

    IMPORTANCE Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

  • An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor [Structure and Assembly]

  • During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage 29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of 29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although 29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages.

    IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage 29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs.

  • Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket [Vaccines and Antiviral Agents]

  • The peptide drug enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but it easily induces drug resistance, calling for new strategies for developing effective drugs. On the basis of the M-T hook structure, we recently developed highly potent short-peptide HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the conserved gp41 pocket and possess high genetic barriers to resistance. Here, we focused on the selection and characterization of HIV-1 escape mutants of MTSC22, which revealed new resistance pathways and mechanisms. Two mutations (E49K and L57R) located at the inhibitor-binding site and two mutations (N126K and E136G) located at the C-terminal heptad repeat region of gp41 were identified as conferring high resistance either singly or in combination. While E49K reduced the C-terminal binding of inhibitors via an electrostatic repulsion, L57R dramatically disrupted the N-terminal binding of M-T hook structure and pocket-binding domain. Unlike E49K and N126K, which enhanced the stability of the endogenous viral six-helical bundle core (6-HB), L57R and E136G conversely destabilized the 6-HB structure. We also demonstrated that both primary and secondary mutations caused the structural changes in 6-HB and severely impaired the capability for HIV-1 entry. Collectively, our data provide novel insights into the mechanisms of short-peptide fusion inhibitors targeting the gp41 pocket site and help increase our understanding of the structure and function of gp41 and HIV-1 evolution.

    IMPORTANCE The deep pocket on the N-trimer of HIV-1 gp41 has been considered an ideal drug target because of its high degree of conservation and essential role in viral entry. Short-peptide fusion inhibitors, which contain an M-T hook structure and mainly target the pocket site, show extremely high binding and inhibitory activities as well as high genetic barriers to resistance. In this study, the HIV-1 mutants resistant to MTSC22 were selected and characterized, which revealed that the E49K and L57R substitutions at the inhibitor-binding site and the N126K and E136G substitutions at the C-terminal heptad repeat region of gp41 critically determine the resistance phenotype. The data provide novel insights into the mechanisms of action of the M-T hook structure-based fusion inhibitors which will help further our understanding of the structure-function relationship of gp41 and molecular pathways of HIV-1 evolution and eventually facilitate the development of new anti-HIV drugs.

  • Cocirculation of Two env Molecular Variants, of Possible Recombinant Origin, in Gorilla and Chimpanzee Simian Foamy Virus Strains from Central Africa [Genetic Diversity and Evolution]

  • Simian foamy virus (SFV) is a ubiquitous retrovirus in nonhuman primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory has identified more than 50 hunters from central Africa infected with zoonotic SFVs. Analysis of the complete sequences of five SFVs obtained from these individuals revealed that env was the most variable gene. Furthermore, recombinant SFV strains, some of which involve sequences in the env gene, were recently identified. Here, we investigated the variability of the env genes of zoonotic SFV strains and searched for possible recombinants. We sequenced the complete env gene or its surface glycoprotein region (SU) from DNA amplified from the blood of (i) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee foamy virus (FV) strain and (ii) 1 gorilla and 3 infected chimpanzees living in the same areas as these hunters. Phylogenetic analyses revealed the existence of two env variants among both the gorilla and chimpanzee FV strains that were present in zoonotic and NHP strains. These variants differ greatly (ggt;30% variability) in a 753-bp-long region located in the receptor-binding domain of SU, whereas the rest of the gene is very conserved. Although the organizations of the Env protein sequences are similar, the potential glycosylation patterns differ between variants. Analysis of recombination suggests that the variants emerged through recombination between different strains, although all parental strains could not be identified.

    IMPORTANCE SFV infection in humans is a great example of a zoonotic retroviral infection that has not spread among human populations, in contrast to human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). Recombination was a major mechanism leading to the emergence of HIV. Here, we show that two SFV molecular envelope gene variants circulate among ape populations in Central Africa and that both can be transmitted to humans. These variants differ greatly in the SU region that corresponds to the part of the Env protein in contact with the environment. These variants may have emerged through recombination between SFV strains infecting different NHP species.

  • Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity [Structure and Assembly]

  • The membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (+1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this +1 leucine mutant by either inserting two additional amino acids (+3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the +1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the +1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network.

    IMPORTANCE The membrane-proximal external region (MPER) and membrane-spanning domains (MSDs) of viral glycoproteins have been shown to be critical for regulating glycoprotein fusogenicity. However, the roles of these two domains are poorly understood. We report here that point deletions and insertions within the MPER or MSD result in functionally inactive proteins. However, when the C-terminal tail domain (CTD) is deleted, the majority of the proteins remain functional. The only residues that were found to be critical for function regardless of the CTD were four hydroxyl-containing amino acids located at the C terminus of the MPER (T139 and T140) and at the N terminus of the MSD (S143 and T144) and a proline near the beginning of the MSD (P148). We demonstrate that hydrogen-bonding at positions T139 and S143 is critical for protein function. Our findings provide novel insights into the role of the MPER in regulating fusogenic activity of viral glycoproteins.

  • Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization [Vaccines and Antiviral Agents]

  • The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 x PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 mmu;g/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans.

    IMPORTANCE To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.

  • Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain [Virus-Cell Interactions]

  • Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe viruses, which are not known to naturally cause human diseases. Using a recombinant Pichinde virus system, we show that the LCMV Z N-terminal domain (NTD) mediates the inhibition of macrophage activation and immune functions.

  • AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4+ T Cells [Virus-Cell Interactions]

  • HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, bbeta;-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4+ T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.

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    JVI Accepts: Articles Published Ahead of Print

  • Pseudorabies virus US3 protein kinase protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a [PublishAheadOfPrint]

  • Several reports have indicated that Natural Killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse NK cell evasion mechanisms, although only few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The anti-viral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surface of virus-infected cells. Here, we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties towards NK cells by triggering binding of the inhibitory NK receptor CD300a to the infected cell surface, thereby providing increased CD300a-mediated protection of infected cells against NK-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid CD300a ligands and on group I p21-activated kinases. These data identify a novel alphaherpesvirus NK evasion strategy and demonstrate for the first time a role for CD300a in regulating NK cell activity upon contact with virus infected target cells.

    IMPORTANCE Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here, we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays previously uncharacterized immune evasion properties towards NK cells. Expression of US3 protects infected cells from NK-mediated lysis via increased binding of the inhibitory NK receptor CD300a. We show that this US3-mediated increased CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAK). The identification of this novel NK evasion strategy may contribute to the design of improved herpesvirus vaccines and may also be of significance for other PAK- and CD300a-modulating viruses and cancer cells.

  • Oral norovirus infection is blocked in mice lacking Peyer's patches and mature M cells [PublishAheadOfPrint]

  • A critical early step in murine norovirus (MNV) pathogenesis is crossing the intestinal epithelial barrier to reach the target cells for replication, i.e., macrophages, dendritic cells, and B cells. Our previous work showed that MNV replication decreases in the intestines of mice conditionally depleted of microfold (M) cells. To define the importance of Peyer's patch (PP) M cells during MNV pathogenesis, we used a model of Balb/c mice deficient in the recombination activation gene (Rag) 2 and common gamma chain (c) (Rag-c-/-), which lack gut-associated lymphoid tissues (GALT), such as Peyer's patches, and mature GP2+ M cells. Rag-c-/- were infected intraperitoneally or per-orally with MNV-1 or CR3 for 24 and 72 hr. Although, intestinal lamina propria of Rag-c-/- mice have a greater frequency of certain MNV target cells (dendritic cells and macrophages) and lack others (B cells), Rag-c-/- and wild-type Balb/c mice showed relatively similar viral loads in the intestine following infection by the intraperitoneal route, which provides direct access to target cells. However, Rag-c-/- mice were not productively infected orally by MNV, a route in which virions must cross the intestinal epithelial barrier. These data are consistent with a model whereby PP M cells are the primary route for MNV to cross the intestinal epithelium of Balb/c mice.

    IMPORTANCE Noroviruses (NoV) are prevalent pathogens that infect their host via the intestine. Identifying key factors important during the initial stages of virus infection in the host may provide novel points of intervention.

    Microfold (M) cells, antigen sampling cells in the intestine, were previously shown to provide a gateway for murine NoV (MNV) into the host but the relative importance of this uptake pathway remained unknown. Here, we show that the absence of gastrointestinal-associated lymphoid structures (GALT) such as Peyer's patches, which contain high numbers of mature M cells, renders Balb/c mice refractory to oral infection with MNV. These findings are consistent with the model that M cells represent the primary route for MNV to cross the intestinal epithelial barrier and infect underlying immune cells during a productive infection.

  • Enhanced immune responses to HIV-1 envelope elicited by a vaccine regimen consisting of priming with Newcastle disease virus expressing HIV gp160 and boosting with gp120 and SOSIP gp140 proteins. [PublishAheadOfPrint]

  • Newcastle disease virus expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in prime-boost combination in guinea pigs to enhance Env-specific humoral and mucosal immune responses. We showed that NDV prime followed by protein boost regimen elicited stronger serum and mucosal Th-1 biased IgG responses and neutralizing antibody responses compared to NDV only immunizations. Additionally, these responses were higher after gp120 compared to SOSIP gp140 protein boost.

  • Epstein-Barr virus miR-BART20-5p suppresses lytic induction by inhibiting BAD-mediated caspase-3-dependent apoptosis [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a human gamma-herpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as yet unknown form of stimulation, lytic gene expression and progeny virus production commences. Thus far, exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction has yet to be elucidated. Previously, we have shown that EBV miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1, as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In the current study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD mediated caspase-3 -dependent apoptosis which would trigger immediate early gene expression.

    IMPORTANCE EBV has an ability to remain latent in host cells including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly by suppressing BAD-induced caspase-3-dependent apoptosis in addition to directly as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

  • Generation of an avian/mammalian rotavirus reassortant using a helpervirus-dependent reverse genetics system [PublishAheadOfPrint]

  • Genetic diversity of Rotavirus A (RVA) strains is in part facilitated by genetic reassortment. Although this process of genome segment exchange has been reported frequently among mammalian RVAs, it remained elusive if mammalian RVAs can also package genome segments from avian RVA strains. We generated a simian RVA strain SA11 reassortant containing the VP4 gene of chicken RVA strain 02V0002G3. To achieve this, we transfected BSR5/T7 cells with a T7 polymerase-driven VP4-encoding plasmid, infected the cells with a temperature-sensitive SA11 VP4 mutant and selected the recombinant virus by increasing the temperature. The reassortant virus could be stably passaged and exhibited cytopathic effects in MA-104 cells, but replicated less efficient than both parental viruses. Our results show that avian and mammalian rotaviruses can exchange genome segments, resulting in replication-competent reassortants with new genomic and antigenic features.

    IMPORTANCE The study shows that rotaviruses of mammals can package genome segments from rotaviruses of birds. By this, the genetic diversity of rotaviruses could be broadened, which might be important for their antigenic variability. The reverse genetics system applied in the study could be useful for targeted generation and subsequent characterization of distinct rotavirus reassortant strains.

  • Genetics, receptor binding, replication and transmission in mammals of H4 avian influenza viruses isolated from live poultry markets in China [PublishAheadOfPrint]

  • H4 avian influenza virus (AIV) is one of the most prevalent influenza subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to aalpha;-2,6-linked glycans, although they retained the binding preference for aalpha;-2,3-linked glycans. When we tested the direct contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs.

    IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are co-circulating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains can also transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health.

  • A single amino acid change in the Marburg virus matrix protein VP40 provides a replicative advantage in a species-specific manner [PublishAheadOfPrint]

  • Marburg virus (MARV) induces severe hemorrhagic fever in humans and non-human primates but only transient non-lethal disease in rodents. Previous studies demonstrated that sequential passages of MARV in rodents boosts infection to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 (rMARVVP40(D184N)) grew to higher titers than wild type MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN) antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling.

    IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing of how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLPs assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.

  • Machupo virus expressing GPC of the Candid#1 vaccine strain of Junin virus is highly attenuated and immunogenic [PublishAheadOfPrint]

  • Machupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that MACV with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent for Argentine hemorrhagic fever. Others and our group have identified the glycoprotein as the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication, and its ability to attenuate MACV in vivo. Recombinant MACV with Candid#1-glycoprotein (rMACV/Cd#1-GPC) exhibited similar growth properties to Candid#1 and was genetically stable in vitro. In a lethal mouse model, the rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV expressing glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model.

    IMPORTANCE Currently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrated for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.

  • Dysregulation of TLR7 compromises innate and adaptive T cell responses and host resistance to an attenuated West Nile virus infection in old mice [PublishAheadOfPrint]

  • The elderly are known to have enhanced susceptibility to infections and impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6-to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21-to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection, but were protected from subsequent lethal wild- type WNV challenge. Compared to young mice, NS4B-P38G mutant triggered higher inflammatory cytokine and IL-10 production, a delayed T cell expansion, and lower antibody and WNV- specific T cell responses in old mice. Toll-like receptor (TLR) 7 is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen presenting function compromise, and a reduced T cell and Treg expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist decreased host vulnerability in NS4B-P38G infected old mice by enhancing T cell and Treg expansion and antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with WNV NS4B-P38G mutant.

    IMPORTANCE:The elderly are known to have enhanced susceptibility to infections and impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

  • RNA replication and membrane modification require the same functions of alphavirus nonstructural proteins [PublishAheadOfPrint]

  • The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants, including those defective in RNA capping, capable of negative-strand synthesis, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

  • HIV-1 monoclonal antibodies suppress acute SHIV viremia and limit seeding of cell-associated viral reservoirs [PublishAheadOfPrint]

  • Combination antiretroviral therapy (cART) administered shortly after HIV-1 infection can suppress viremia and limit seeding of the viral reservoir, but lifelong treatment is required in the majority of patients. Highly potent broadly neutralizing HIV-1 monoclonal antibodies (mAbs) can reduce plasma viremia when administered during chronic HIV-1 infection, but the therapeutic potential of these antibodies during acute infection is unknown. We tested the ability of HIV-1 envelope glycoprotein specific broadly neutralizing mAbs to suppress acute simian-human immunodeficiency virus (SHIV) replication in rhesus macaques. Four groups of macaques were infected with SHIV-SF162P3 and received i) the CD4-binding site mAb VRC01 or ii) a combination of a more potent clonal relative of VRC01 (VRC07-523) and a V3 glycan-dependent mAb (PGT121) or iii) daily cART, all on day 10, just prior to expected peak plasma viremia, or iv) no treatment. Daily cART was initiated 11 days after mAb administration and was continued for 13 weeks in all treated animals. Over a period of 11 days after a single administration, mAb treatment significantly reduced peak viremia, accelerated the decay slope and reduced total viral replication as compared to untreated controls. Proviral DNA in lymph node CD4 T cells was also diminished after treatment with dual mAb. These data demonstrate the virological effect of potent mAbs and support future clinical trials that investigate HIV-1 neutralizing mAbs as adjunctive therapy with cART during acute HIV-1 infection.

    IMPORTANCE Treatment of chronic HIV-1 infection with potent broadly neutralizing HIV-1 mAbs has been shown to significantly reduce plasma viremia. However, the anti-viral effect of mAb treatment during acute HIV-1 infection is unknown. Here, we demonstrate that mAbs targeting the HIV-1 envelope glycoprotein both suppress acute SHIV plasma viremia and limit CD4 T cell-associated viral DNA. These findings provide support for clinical trials of mAbs as adjunctive therapy with antiretroviral therapy during acute HIV-1 infection.

  • Knockdown of autophagy inhibits infectious hepatitis C virus release by exosomal pathway [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in humans. We have shown earlier that HCV induces autophagy for viral persistence by preventing innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism for the observation remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCV infected cells. We observed an association between HCV and exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that extracellular infectious HCV is significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an IFN-stimulated gene BST-2 is upregulated in autophgy knockdown cells and associated with exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes.

    IMPORTANCE Autophagy plays an important role in HCV pathogenesis. Autophagy suppresses innate immune response and promotes survival of virus infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosome to lysosome, thus providing a link with exosome. Inhibition of HCV induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

  • Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures [PublishAheadOfPrint]

  • All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cultures. Because HAE cultures are isolated directly from human airways, Vero-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore require a larger inoculum for effective vaccination. We hypothesized that Vero-derived virus containing an intact G protein would be more infectious for HAE cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L as the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero-grown virions, but not in HeLa-grown virions suggesting a difference in G protein posttranslational modification in the two cell lines. We identified amino acids important for cleavage by mutagenesis, and these amino acids include a likely cathepsin L cleavage site. Virus containing a modified, non-cleavable G protein, produced in Vero cells was 5-fold more infectious for HAE cultures, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines.

    IMPORTANCE Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein are less infectious for primary airway epithelial cells, the natural RSV target. Here we identify the protease responsible, locate the cleavage site, and demonstrate that cleavage likely occurs during endocytic recycling. Moreover, we show that Vero-derived viral infectivity is increased 5-fold for primary airway epithelial cells if the virus contains a mutation in the G protein that prevents cleavage. Blocking cleavage should improve RSV vaccine yield, consequently reducing production costs. Posttranslational cleavage of the fusion glycoprotein of many viruses plays an essential role in activation, however, cleavage of the RSV G protein is a novel example of a detrimental effect of cleavage on virus infectivity.

  • The structure of Human Parechovirus-1 reveals an association of the RNA genome with the capsid [PublishAheadOfPrint]

  • Parechoviruses are human pathogens that cause diseases ranging from gastrointestinal disorders to encephalitis. Unlike most picornaviruses, parechovirus capsids are composed of only three subunits: VP0, VP1, and VP3. Here we present the structure of a human parechovirus-1 (HPeV-1) virion determined to a resolution of 3.1 AAring;. We find that interactions among pentamers in the HPeV-1 capsid are mediated by the N-termini of VP0s, which correspond to the capsid protein VP4 and the N-terminal part of the capsid protein VP2 of other picornaviruses. In order to facilitate delivery of the virus genome into the cytoplasm, the N-termini of VP0s have to be released from contacts between pentamers and exposed at the particle surface, resulting in capsid disruption. A hydrophobic pocket, which can be targeted by capsid-binding anti-viral compounds in many other picornaviruses, is not present in HPeV-1. However, we found that interactions between the HPeV-1 ssRNA genome and subunits VP1 and VP3 in the virion impose a partial icosahedral ordering on the genome. The residues involved in the RNA binding are conserved among all parechoviruses, suggesting putative role of the genome in virion stability or assembly. Therefore, putative small molecules that could disrupt HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors.

    IMPORTANCE Human Parechoviruses (HPeV) are pathogens that cause diseases ranging from respiratory and gastrointestinal disorders to encephalitis. Recently, there have been outbreaks of HPeV infections in Western Europe and North America. We present the first atomic structure of parechovirus HPeV-1 determined by X-ray crystallography. The structure explains why HPeVs cannot be targeted by anti-viral compounds that are effective against other picornaviruses. Furthermore, we found that the interactions of the HPeV-1 genome with the capsid resulted in a partially icosahedral ordering of the genome. The residues involved in the RNA binding are conserved among all parechoviruses, suggesting an evolutionarily fixed role of the genome in virion assembly. Therefore, putative small molecules disrupting HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors.


  • Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favours expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression.

    In the present study we investigated the Rex-dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild type and Rex-knock out HTLV-1 molecular clones using splice site-specific qRT-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex-dependent, while the p21rex mRNA was Rex-independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2rrsquo; -hydroxyl acylation analysed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to express Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency which might influence the ability of the virus to spread and evade the immune system.

    IMPORTANCE HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favours expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to express Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency which might influence the ability of the virus to spread and evade the immune system.

  • Mechanism of cell culture adaptation of an enteric calicivirus, porcine sapovirus Cowden strain [PublishAheadOfPrint]

  • Porcine sapovirus (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to wild-type (WT) PoSaV Cowden strain, the tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse genetics system, we identified that four (178, 289, 324, and 328) amino acid substitutions in VP1, but not the substitutions in the RdRp region, were critical for the cell culture adaptation of PoSaV Cowden strain. The other two substitutions in VP1 (291 and 295) reduced virus replication in vitro. Three dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at the protruding subdomain 2 (P2) of VP1, which may influence virus binding to the cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutations at residues 291 or 295 from that of TC to WT reduced virus replication in vitro, it enhanced viral replication in vivo, and the revertants induced higher serum and mucosal antibody responses compared to TC PoSaV Cowden strain. Our findings have revealed the molecular basis for PoSaV adaptation to cell culture. They may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.

    IMPORTANCE Tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that four amino acid substitutions in VP1 (178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (291 and 295) reduced virus replication in vitro, but enhanced virus replication and induced higher serum and mucosal antibody responses in gnotobiotic pigs, compared with the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization, residues 289, 291, 324, and 328 may influence virus binding to the cellular receptors, and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

  • Synonymous deoptimization of the foot-and-mouth disease virus causes attenuation in vivo while inducing a strong neutralizing antibody response. [PublishAheadOfPrint]

  • Codon bias deoptimization has been previously used to successfully attenuate human pathogens including polio, respiratory syncytial and influenza viruses. We have applied a similar technology to deoptimize the capsid coding region (P1) of foot-and-mouth disease virus (FMDV). Despite the introduction of 489 nucleotide changes (19%), synonymous deoptimization of the P1 region rendered a viable FMDV progeny. The resulting strain was stable and reached cell culture titers similar to those obtained for wild type (WT) virus, but at reduced specific infectivity. Studies in mice showed that 100% of animals inoculated with FMDV A12 P1 deoptimized mutant (A12-P1 deopt) survived, even when the animals were infected at doses 100 times higher than the dose required to cause death by WT virus. All mice inoculated with A12-P1 deopt mutant developed a strong antibody response and were protected against subsequent lethal challenge with WT virus at 21 days post inoculation. Remarkably, vaccine safety margin was at least 1,000 fold higher for A12-P1 deopt as compared to WT virus. Similar patterns of attenuation were observed in swine, in which animals inoculated with A12-P1 deopt virus did not develop clinical disease until doses reached 1,000-10,000 times the dose required to cause severe disease in two days with A12 WT. Consistently, high levels of antibody titers were induced, even at the lowest dose tested. These results highlight the potential use of synonymous codon pair deoptimization as a strategy to safely attenuate FMDV and further develop live attenuated vaccine candidates to control such a feared livestock disease.

    IMPORTANCE Foot-and-mouth disease (FMD) is one of the most feared viral diseases that can affect livestock. Although this disease appeared to be contained in developed nations by the end of the last century, recent outbreaks in Europe, Japan, Taiwan, South Korea, etc, have demonstrated that infection can spread like wild-fire causing devastating economic and social consequences. The Global Foot-and-Mouth Disease Research Alliance (GFRA), an international organization launched in 2003, has set as part of their five main goals, the development of next generation control measures and strategies including improved vaccines and biotherapeutics. Our work demonstrates that newly developed codon pair bias deoptimization technologies can be applied to FMD virus to obtain attenuated strains with potential for further development as novel live attenuated vaccine candidates that may rapidly control disease without reverting to virulence.

  • Host miR-197 plays a negative regulatory role in the enterovirus 71 infectious cycle by targeting the RAN protein [PublishAheadOfPrint]

  • Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3rrsquo; UTR reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication.

    IMPORTANCE Enterovirus 71 (EV71) is a picornavirus with (+) single-stranded RNA that globally inhibits the cellular translational system mainly by cleaving cellular eIF4G and PABP, which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target-site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.

  • Origin of Rebound Plasma HIV Includes Cells with Identical Proviruses that are Transcriptionally Active Before Stopping Antiretroviral Therapy [PublishAheadOfPrint]

  • Understanding the origin of HIV variants during viral rebound may provide insight into the composition of the HIV reservoir and has implications for the design of curative interventions. HIV single-genome sequences were obtained from ten AIDS Clinical Trials Group participants who underwent analytic ART interruption (ATI). Rebounding variants were compared with those in pre-ART plasma in all 10 participants and with on-ART PBMC-associated DNA and RNA (DNA and CA-RNA) in 7/10 participants. The highest viral diversities were found in the DNA and CA-RNA populations. In 3 of 7 participants, we detected multiple, identical DNA and CA-RNA sequences during suppression on ART that exactly matched rebounding plasma HIV sequences. Hypermutated DNA and CA-RNA were detected in four participants, contributing to diversities in these compartments that were higher than in the pre-ART and post-ART plasma. Shifts in the viral rebound populations could be detected in some participants over the 2-3 month observation period. These findings suggest that a source of initial rebound viremia could be populations of infected cells that clonally expanded either prior to and/or during ART, some of which were already expressing HIV RNA before treatment was interrupted. These clonally-expanding populations of HIV-infected cells may represent an important target for strategies aimed at achieving reservoir reduction and sustained virologic remission.

    IMPORTANCE Antiretroviral therapy alone cannot eradicate the HIV reservoir and viral rebound is generally rapid after treatment interruption. It has been suggested that clonal expansion of HIV-infected cells is an important mechanism of HIV reservoir persistence, but the contribution of these clonally-proliferating cells to the rebounding virus is unknown. We report a study of AIDS Clinical Trials Group participants who underwent treatment interruption and compared rebounding plasma virus with that found within cells prior to treatment interruption. We found several incidences where plasma HIV variants exactly matched that of multiple proviral DNA copies from infected blood cells sampled before treatment interruption. In addition, we found that these cells were not dormant, but were generating unspliced RNA transcripts before treatment was interrupted. Identification of the HIV reservoir and determining its mechanisms for persistence may aid in the development of strategies towards a cure for HIV.

  • A haploid genetic screen identifies heparan sulfate proteoglycans supporting Rift Valley fever virus infection [PublishAheadOfPrint]

  • Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1 deficient cells exhibit altered Golgi morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors.

    IMPORTANCE Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents, and details of its viral life cycle and interaction with host cells are not well characterized. We used the power of genetic screening in human cells and found that RVFV utilizes glycosaminoglycans to attach to host cells. This furthers our understanding of the virus and informs development of antiviral therapeutics.

  • Identification of Viral and Host Proteins that Interact with MHV68 Latency-Associated Nuclear Antigen during Lytic Replication: A Role for Hsc70 in Viral Replication [PublishAheadOfPrint]

  • Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus-68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through co-immunoprecipitation and in vitro GST pulldown assays. Immunofluorescence and cellular fractionation analyses comparing WT to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively-infected cells. Pharmacologic inhibition and shRNA-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc70 in facilitating MHV68 lytic replication.

    IMPORTANCE Latency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68 this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat-shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral replication.

  • Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Co-option and Domestication [PublishAheadOfPrint]

  • Endogenous retroviruses (ERVs) are remnants of ancestral retroviral infections of germ cells. Retroviral endogenization is an adaptation process for the host genome, and ERVs are gradually attenuated or inactivated by mutation. However, some ERVs that have been "domesticated" by their hosts eventually gain physiological functions, such as placentation or viral resistance. We previously reported the discovery of Refrex-1, a soluble anti-retroviral factor in domestic cats that specifically inhibits infection by Feline leukemia virus subgroup D (FeLV-D), a chimeric virus of FeLV and a feline ERV, ERV-DC. Refrex-1 is a truncated envelope protein (Env) encoded by both ERV-DC7 and ERV-DC16 proviral loci. Here, we reconstituted ancestral and functional Env from ERV-DC7 and ERV-DC16 envelope genes (env) by inducing reverse mutations. Unexpectedly, ERV-DC7 and ERV-DC16 full-length Env (ERV-DC7 fl and ERV-DC16 fl), reconstructed by removing stop codons, did not produce infectious viral particles. ERV-DC7 fl and ERV-DC16 fl were highly expressed in cells, but were not cleaved into surface subunits (SU) and transmembrane subunits, nor were they incorporated into virions. G407R/N427I-A429T and Y431D substitutions within the SU C-terminal domain of ERV-DC7 fl and ERV-DC16 fl, respectively, caused these dysfunctions. The 407 glycine and 431 tyrosine residues are relatively conserved among infectious gammaretroviruses, and their substitution causes the same dysfunctions as the tested retroviruses. Our results reveal that specific mutations within the SU C-terminal domain suppressed Env cleavage and incorporation into virions and indicate that these mutations contributed to the domestication of Refrex-1 through multi-step events that occurred in the post-integration period.

    IMPORTANCE Domestic cats are colonized with various exogenous retroviruses (exRVs), such as feline leukemia virus (FeLV), and their genomes contain numerous ERVs, some of which are replication-competent proviruses. The feline hosts, exRVs, and ERVs have complicated genetic interactions, and provide an interesting field model for triangular relationships: recombination between FeLV and ERV-DC, which is a feline ERV, generated FeLV-D, a chimeric virus, and FeLV-D is restricted by Refrex-1, an anti-retroviral factor corresponding to truncated Env of ERV-DC7 and ERV-DC16. Here, we reconstructed ancestral, functional Env from ERV-DC7 and ERV-DC16 env by inducing reverse mutations to elucidate how Refrex-1 was generated from its ancestor. Our results reveal that they were repeatedly inactivated by mutations preventing Env maturation. Our results provide insights into how ERVs were "domesticated" by their hosts and identify the mutations that mediated these evolutions. Notably, experiments that restore inactivated ERVs might uncover previously unrecognized features or properties of retroviruses.

  • Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells [PublishAheadOfPrint]

  • Tetherin (BST2, CD317 or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three-dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in similar positions to the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane, but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions were often arranged in a linear fashion, primarily as single chains and to a lesser degree as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes.

    IMPORTANCE Tetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ~15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

  • The C5-substituted Tetrahydropyrano-tetrahydofuran-derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs Including Darunavir [PublishAheadOfPrint]

  • We identified three non-peptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097 containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT) with 50% effective concentrations (EC50s) of 3.0-49 nM and minimal cytotoxicity with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, ggt;100, and ggt;100 mmu;M, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multi-drug resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was ggt;1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVRP51), the three compounds remained active to HIV-1DRVRP51 only with 6.8- to 68-fold reduction. Moreover, the emergence of drug resistant HIV-1s against the three compounds was considerably delayed compared to the case of DRV. Especially, HIV-1 variants resistant to GRL-085 and -097 did not emerge even by using two different highly-DRV-resistant HIV-1s as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen-bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings warrant that the three compounds be further studied as possible therapeutic agents for treating individuals harboring wild-type and/or HIVMDR.

    IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1s and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1s (HIVDRVR) has recently been reported in vivo and in vitro. Here we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C5 hydroxyl (GRL-015, -085, and -097), which potently suppress the replication of HIVDRVR. Moreover, the emergence of drug resistant HIV-1s against the three compounds was considerably delayed compared to the case of DRV. The C5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased the activity against HIVDRVRs. The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRVR.

  • Engineering Enhanced Vaccine Cell Lines to Eradicate Vaccine-Preventable Diseases: The Polio Endgame [PublishAheadOfPrint]

  • Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNAi screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers ggt;20-fold and ggt;50-fold, respectively. Host gene knockdown events did not affect virus antigenicity and CRISPR-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced enterovirus 71, a clinically relevant virus for which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing and should facilitate polio eradication using the inactivated poliovirus vaccine.

    IMPORTANCE Using a genome-wide RNAi screen, a collection of host virus-resistance genes was identified that upon silencing increased poliovirus and enterovirus 71 production from 10-fold to ggt;50-fold in a Vero vaccine manufacturing cell line. This study provides novel insights into enterovirus-host interactions, and describes an approach toward developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The findings show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin) and wild polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine, as well as further reducing costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine preventable diseases.

  • A Comprehensive RNA-Seq Analysis of the Adeno-Associated Virus type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins [PublishAheadOfPrint]

  • Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on co-infection with adenovirus (Ad), and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon co-infection with Ad. So far, AAV transcripts have only been characterized on the (+) strand of the AAV ssDNA genome. The AAV (-) strand is assumed not to be transcribed. Here we apply Illumina-based RNA-Seq to characterize the entire AAV2 transcriptome in the absence and presence of Ad. We find known- and identify novel AAV transcripts including additional splice variants, the most abundant of which leads to expression of a novel 18 kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV (-) strand with clustered reads upstream of the p5 promoter, confirmed by 5rrsquo; RACE and RNase protection assays. The p5 promoter displays considerable activity in both directions, indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon co-infection with Ad.

    IMPORTANCE Next generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18 kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV (-) strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV in vivo, combined in cellulae and in silico studies will help to forward the understanding of the unique, bipartite AAV life cycle.

  • Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is the major viral cause of birth defects, a serious problem in immunocompromised individuals, and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates, but not in mammals, and induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit the HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease.

    IMPORTANCE HCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the non-toxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of developing resistance to treatment.

  • Occupancy of RNA Polymerase II (S5P) and RNA Polymerase II (S2P) on VZV Genes 9, 51 and 66 is Independent of Transcript Abundance and Polymerase Location within the Gene [PublishAheadOfPrint]

  • Regulation of varicella zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, gene transcription requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and occupancy phosphorylated RNA polymerase II (RNAP) on the gene promoter, body and terminus of VZV genes 9, 51 and 66. The abundance of VZV gene 9, 51 and 66 transcripts in VZV-infected HFL cells was determined by reverse transcription linked qPCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5P) at VZV genes 9, 51 and 66 independent of transcript abundance and location within the virus gene at both 1 and 3 days post infection (dpi). In contrast, serine 2 (S2P) modified RNAP was not detected at any virus gene location at 3 dpi, and only slightly above background levels at 1 dpi.

    IMPORTANCE Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromosomal immunoprecipitation (ChIP). We have used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that the C-terminal domain of RNAP hyperphosphorylated at serine 5 along the lengths of VZV genes (promoter, body and transcriptional termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation RNAP was detected at these virus gene regions. This is distinct from RNAP at highly regulated host genes, where RNAP S5P occupancy decreases and S2P levels increase as the polymerase transits through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.

  • Minor Contribution of Host-HIV Readthrough Transcripts to the Level of HIV Cell-associated gag RNA [PublishAheadOfPrint]

  • Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor the reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but chimeric host-HIV readthrough transcripts. Here we demonstrate that in HIV-infected patients on suppressive cART, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

  • Human Cytomegalovirus modulates Expression of non-canonical Wnt receptor ROR2 to Alter Trophoblast Migration [PublishAheadOfPrint]

  • Maternal primary Cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus, but also from indirect effects through placental infection and dysfunction. CMV alters Wingless (Wnt) signalling, an essential cellular pathway involved in placentation, as evidenced by reduced transcription of canonical Wnt target genes and decreased Wnt3a-induced trophoblast migration. Whether CMV affects the non-canonical Wnt signalling pathway has been unclear. This study demonstrates for the first time that CMV infection inhibits Wnt5a-stimulated migration of human SGHPL-4 trophoblasts, and that inhibition of the pathway restores normal migration of CMV-infected cells. Western blot and real-time PCR analyses show increased expression of non-canonical Wnt receptor ROR2 in CMV-infected trophoblasts. Mimicking the CMV-induced ROR2 protein expression via ectopic expression inhibited Wnt5a-induced trophoblast migration and reduced TCF/LEF-mediated transcription as measured using luciferase reporter assays. Gene silencing using siRNA duplexes decreased ROR2 transcript and protein levels. In contrast, proliferation of SGHPL-4 trophoblasts, measured by MTT assay was not affected. The siRNA-mediated down regulation of ROR2 in trophoblasts rescued CMV-induced reduction in trophoblast migration. These data suggest a mechanism where CMV alters the expression of the Wnt receptor ROR2 to alter Wnt5a-mediated signalling and inhibit trophoblast motility. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage, and consequent fetal damage in congenital CMV infections.

    IMPORTANCE Maternal primary Cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus, but also from indirect effects through placental infection and placental dysfunction. No effective therapy is currently proven to prevent or treat congenital CMV infection. Understanding the molecular underpinnings of CMV infection of the placenta is essential for therapeutic innovations and vaccine design. CMV alters canonical Wingless (Wnt) signalling, an essential cellular pathway involved in placental development. This study suggests a mechanism where CMV alters the expression of non-canonical Wnt receptor ROR2 to alter motility of placental cells, which has important implications in the pathogenesis of CMV-induced placental dysfunction. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage, and consequent fetal damage in congenital CMV infection.

  • Epstein-Barr Virus gp350 Can Functionally Replace the Rhesus Lymphocryptovirus Major Membrane Glycoprotein and Does Not Restrict Infection of Rhesus Macaques [PublishAheadOfPrint]

  • Primary Epstein-Barr virus (EBV) infection is the most common cause of infectious mononucleosis, and persistent infection is associated with multiple cancers. EBV vaccine development has focused on the major membrane glycoprotein, gp350, since it is the major target for antibodies that neutralize infection of B cells. However, EBV has a tropism for both B cells and epithelial cells, and it is unknown whether serum neutralizing antibodies against B cell infection will provide sufficient protection against virus infection initiated at the oral mucosa. This could be stringently tested by passive antibody transfer and oral virus challenge in the rhesus macaque model for EBV infection. However, only neutralizing monoclonal antibodies (mabs) against EBV are available, and EBV is unable to infect rhesus macaques because of a host range restriction of unknown mechanism. We cloned the prototypic EBV neutralizing antibody, 72A1, and found that recombinant 72A1 did not neutralize rhLCV infection of macaque B cells. Therefore, we constructed a chimeric rhesus lymphocryptovirus (rhLCV) where the native major membrane glycoprotein was replaced with EBV gp350. This chimeric rhLCV became sensitive to neutralization by the 72A1 mab, efficiently immortalized macaque B cells in vitro, and successfully established acute and persistent infection after oral inoculation of rhesus macaques. Thus, EBV gp350 can functionally replace rhLCV gp350 and does not restrict rhLCV infection in vitro or in vivo. The chimeric rhLCV enables direct use of an EBV-specific mab to investigate the effects of serum neutralizing antibodies against B cell infection on oral viral challenge in rhesus macaques.

    IMPORTANCE This study asked whether the EBV major membrane glycoprotein could functionally replace the rhLCV major membrane glycoprotein. We find that a rhLCV humanized with EBV gp350 is capable of efficiently immortalizing monkey B cells in vitro and reproduces acute and persistent infection after oral inoculation of macaques. These results advance our understanding of why EBV cannot infect rhesus macaques by proving that viral attachment through gp350 is not the mechanism for EBV host range restriction. Humanization of rhLCV with EBV gp350 also confers susceptibility to a potent EBV neutralizing mab and provides a novel and significant enhancement to the rhesus macaque animal model where both the clinical utility and biological role of neutralizing mabs against B cell or epithelial cell infection can now be directly tested in the most accurate animal model for EBV infection.

  • Crystal structure of the core region of hantavirus nucleocapsid protein reveals the mechanism for ribonucleoprotein complex formation [PublishAheadOfPrint]

  • Hantaviruses, which belong to the Hantavirus genus of the Bunyaviridae family, infect mammalian animals and humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domain of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcores exclude the N- and C-terminal portions of full polypeptide to get stable proteins for crystallographic study. The structure features an N-lobe and a C-lobe to clamp RNA-binding crevice, and presents two protruding extensions in both lobes. The positively charged residues located in RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, EM visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist amongst bunyaviruses.

    IMPORTANCE: Hantaviruses distribute in wide and increasing range of host reservoirs throughout the world. Particularly, hantaviruses can be transmitted via aerosols of rodent excreta to humans or human-to-human and cause HFRS and HCPS with mortalities of 15% and 50%, respectively. Hantaviruses are therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity, and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection.

  • HIV-1 virion production from single inducible proviruses following T-cell activation ex vivo [PublishAheadOfPrint]

  • Quantifying induced virion production from single proviruses is important for assessing the effects HIV-1 latency reversal agents. Limiting dilution ex vivo cultures of resting CD4+T-cells from 14 HIV-positive volunteers revealed that virion production after T-cell activation from individual proviruses varies by 10,000 to 100,000-fold. High-producing proviruses were associated with increases in cell-associated HIV-1 DNA, suggesting that reactivated proviruses proliferate. Single-cell analyses are needed to investigate variation in proviral expansion and virus production following latency reversal.

  • A cell-free assembly system for generating infectious Human Papillomavirus Type 16 capsids implicates a size discrimination mechanism for preferential viral genome packaging [PublishAheadOfPrint]

  • We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free in vitro assembled particles required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing a llt;8 kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size-discrimination mechanism for papillomavirus genome packaging, in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure.

    IMPORTANCE Little is known about papillomavirus assembly biology due to the difficulties to propagate virus in vitro. The cell-free assembly method established on this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for anti-viral therapy, and allow for the development of new gene delivery systems based on in vitro generated papillomavirus vectors.

  • Human herpesvirus 6A U14 is important for virus maturation [PublishAheadOfPrint]

  • Human herpesvirus 6A U14 is a virion protein of little known function in virus propagation. Here, we elucidated its function by constructing and analyzing U14-mutated viruses. We found that U14 is essential for HHV-6A propagation. We then constructed a mutant virus harboring dysfunctional U14. This virus showed severely reduced growth and retarded maturation. Taken together, these data indicate that U14 plays an important role during HHV-6A maturation.

  • Herpes Simplex Virus Type 1 (HSV-1) and HSV-2 Mediate Species Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1 [PublishAheadOfPrint]

  • Receptor-interacting kinase3(RIP3) and its substrate MLKL (mixed lineage kinase domain like protein) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defensive mechanism. Here, we report that human herpes simplex virus type 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of a RIP1/RIP3 necrosome. HSV R1 was sufficient to suppress TNF-induced necrosis and its RHIM domain was required to disrupt RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defensive mechanism of programmed necrosis in human cells.

    IMPORTANCE This study demonstrated that HSV-1 or HSV-2 infection blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of RIP1/RIP3 complex. This study provides new insights into the species specific modulation of programmed necrosis by HSV.

  • Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative EBV SM Protein [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple post-transcriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effect of SM on all EBV transcripts by high-throughput RNA sequencing, qPCR and Northern blotting. The effect of SM on EBV mRNAs was highly skewed, and was most evident on thirteen late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those which are intrinsically unstable and poorly expressed.

    IMPORTANCE This study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability.

  • Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Boosts Hemagglutinin Stalk-Specific Antibodies [PublishAheadOfPrint]

  • Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of pre-existing, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza infected ferrets were protected from challenge with novel H1N1 influenza virus (CA/09) and these ferrets poorly transmitted the virus to naïve contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus.

    IMPORTANCE: The influenza hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacity, these antibodies are largely strain-specific and do not confer protection against antigenic drift variant or novel HA subtype expressing viruses. By contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity amongst viruses within and among influenza subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naïve contacts.

  • Population-level Immune-mediated Adaptation in HIV-1 Polymerase during the North American Epidemic [PublishAheadOfPrint]

  • Human Leukocyte Antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 Polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979-1989, N=338) and modern (2001-2011, N= 278) eras from across North America (Vancouver, Boston, New York, and San Francisco). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence near-identical to the modern North American subtype B consensus. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold higher patristic (tip-to-tip) genetic distances compared to historic ones, with HLA pressures likely driving ongoing diversification. Moreover, the frequency of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ~2.5-fold higher in modern compared to historic eras, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. Overall however, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 Polymerase sequence substantially pre-adapted to their HLA profile, even in the present era.

    IMPORTANCE HLA class I-restricted CTL escape mutations in HIV-1 that persist upon transmission may accumulate in circulation over time, potentially undermining host antiviral immunity to the transmitted viral strain. We studied ggt;600 experimentally-collected HIV-1 Polymerase sequences linked to host HLA information dating back to 1979, along with phylogenetically reconstructed HIV-1 sequences dating back to the virus' introduction into North America. Overall, our results support the gradual spread of many - though not all - HIV-1 immune escape mutations in circulation over time. This is consistent with recent observations from other global regions, though the extent of polymorphism accumulation in North America appears lower than in populations with high seroprevalence, older epidemics, and/or limited HLA diversity. Importantly, the risk of acquiring an HIV-1 Polymerase sequence at transmission that is substantially pre-adapted to one's HLA profile remains relatively low in North America, even in the present era.

  • RNA-seq Revealed Pyroptosis-induced CD4+ T Cell Death in Early SIV Infected Lymphoid Tissues [PublishAheadOfPrint]

  • Lymphoid tissues (LTs) are principal sites where HIV-1 replicates and virus-host interactions take place, resulting in immunopathology in the form of inflammation, immune activation, and CD4+ T cell death. The HIV-1 pathogenesis in LTs has been extensively studied, however, the understanding of the virus-host interactions in the very early stages of infection remains incomplete. We investigated virus-host interactions in the rectal draining lymph nodes (dLNs) of rhesus macaques at days post intra-rectal inoculation (dpi) with simian immunodeficiency virus (SIV). At 3 dpi, 103 differentially expressed genes (DEGs) were detected using RNA-seq. At 6 and 10 dpi, concomitant with increased SIV replication, 366 and 1350 DEGs were detected, respectively, including up-regulation of genes of innate anti-viral immune responses, inflammation and immune activation. Notably, genes (IFI16, caspase-1, and IL-1bbeta;) in the canonical pyroptosis pathway were significantly up-regulated in expression. We further validated increased pyroptosis using flow cytometry and found that the number of CD4+ T cells expressing activated caspase-1 protein, the hallmark of ongoing pyroptosis, were significantly increased, which is correlated with decreased CD4+ T cells in dLNs. Our results demonstrated that pyroptosis contributes to the CD4+ T cell death in vivo in early SIV infection, suggesting pyroptosis may play a pivotal role in the pathogenesis of SIV, and by extension of HIV-1, since pyroptosis not only induces CD4+ T cell death, but also amplifies inflammation and immune activation. Thus blocking CD4+ T cell pyroptosis could be a complementary treatment to antiretroviral therapy.

    IMPORTANCE Although secondary lymphoid tissues (LTs) are principal sites of HIV-1 replication, inflammation, immune activation and CD4+ T cell death, immunopathogenesis in LTs during early infection remains largely unknown. Using simian immunodeficiency virus (SIV)- rhesus monkey model of HIV rectal infection, we investigated early virus-host interactions. Our results revealed elevated potent host responses in early infection in LTs, including up-regulation of genes involved in anti-viral immune response, inflammation and immune activation. Importantly, genes involved in canonical pyroptosis pathway were significantly up-regulated, and there was a strong correlation between CD4+ T cell decrease and increased number of CD4+ T cells expressing activated caspase-1 protein, demonstrating that pyroptosis contributes to the CD4+ T cell death in vivo in very early SIV infection. Our finding suggests that blocking pyroptosis may be able to damp CD4+ T cell loss during early SIV infection.

  • The C-Terminus of the Core {beta}-Ladder domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion [PublishAheadOfPrint]

  • NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from the Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core bbeta;-ladder domain of the multimeric NS1 structure, and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from Enterovirus-71 (EV71) into the C-terminal end of NS1 could not only be normally released from the infected cells, but also induced dual protective immunity for the host to counteract the lethal challenge of either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C-terminus of the core bbeta;-ladder domain.

    IMPORTANCE The positive-sense RNA genome of mosquito-borne flaviviruses appears to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Herein, we illustrate that the newly identified C-terminal of the core bbeta;-ladder domain in NS1 could be readily inserted into entities such as EV-71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of using JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

  • The cellular protein complex associated with a transforming region of E1A contains c-MYC [PublishAheadOfPrint]

  • The cell transforming activity of HAdV5 E1A is mediated by the N-terminal half of E1A which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400 and pRb family members. Among these protein interactions, the interaction of pRb family proteins with the CR2 region of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning residues 26 to 35 which also interacted with TRRAP and p400. siRNA-mediated depletion of TRRAP reduced c-MYC interaction with E1A while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7 and ATXN7L3 via TRRAP. Acute shRNA-mediated depletion of c-MYC reduced the E1A transforming activity while depletion of ENY2 and MAX did not. These results suggest that c-MYC association with E1A may, at least partially, play a role in the E1A transformation activity, independent of MAX.

    IMPORTANCE The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2 promotes cell proliferation through inactivating the activities of the pRb family proteins is better understood. Our analysis of E1A proteome revealed the presence of the proto-oncoprotein, c-MYC and ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP while ENY2 interacted independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, E1A that may exploit the activity of a cellular oncogene, c-MYC, in addition to inactivation of the tumor suppressor proteins such as pRb.

  • Characterization of recombinant human cytomegaloviruses encoding IE1 mutants L174P and 1-382 reveals that viral targeting of PML bodies perturbs both intrinsic and innate immune responses [PublishAheadOfPrint]

  • PML is the organizer of cellular structures termed nuclear domain 10 (ND10) or PML-nuclear bodies (PML-NBs) that act as key mediators of intrinsic immunity against human cytomegalovirus (HCMV) and other viruses. The antiviral function of ND10 is antagonized by viral regulatory proteins such as the immediate-early protein IE1 of HCMV. IE1 interacts with PML through its globular core domain (IE1CORE) and induces ND10 disruption in order to initiate lytic HCMV infection. Here, we investigate the consequences of a point mutation (L174P) in IE1CORE, which was shown to abrogate the interaction with PML, for lytic HCMV infection. We found that a recombinant HCMV encoding IE1-L174P displays a severe growth defect similar to that of an IE1 deletion virus. Bioinformatic modeling based on the crystal structure of IE1CORE suggested that insertion of proline into the highly alpha-helical domain severely affects its structural integrity. Consistently, L174P mutation abrogates the functionality of IE1CORE and results in degradation of the IE1 protein during infection. In addition, our data provide evidence that IE1CORE as expressed by a recombinant HCMV encoding IE1 1-382 is not only required to antagonize PML-mediated intrinsic immunity, but affects a recently described function of PML in innate immune signaling. We demonstrate a co-regulatory role of PML in type-I and type-II interferon-induced gene expression and provide evidence that upregulation of interferon-induced genes is inhibited by IE1CORE. In conclusion, our data suggest that targeting PML by viral regulatory proteins represents a strategy to antagonize both intrinsic and innate immune mechanisms.

    IMPORTANCE PML nuclear bodies (PML-NBs), which represent nuclear multiprotein complexes consisting of PML and additional proteins, represent important cellular structures that mediate intrinsic resistance against many viruses including human cytomegalovirus (HCMV). During HCMV infection, the major immediate early protein IE1 binds to PML via a central globular domain (IE1CORE) and we have shown previously that this is sufficient to antagonize intrinsic immunity. Here, we demonstrate that modification of PML by IE1CORE not only abrogates intrinsic defense mechanisms, but it also attenuates the interferon response during infection. Our data show that PML plays a novel co-regulatory role in type-I as well as type-II interferon-induced gene expression which is antagonized by IE1CORE. Importantly, our finding supports the view that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to inhibit both intrinsic and innate immune defense mechanisms.

  • An Herpes Simplex Virus 2 (HSV-2) gD Mutant Impaired for Neural Tropism is Superior to HSV-2 gD Subunit Vaccine to Protect Animals from Challenge with HSV-2 [PublishAheadOfPrint]

  • A recent phase 3 trial with soluble herpes simplex virus 2 (HSV-2) glycoprotein D (gD2t) in adjuvant failed to show protection against genital herpes. We postulated that live attenuated HSV-2 would provide more HSV antigens for induction of virus-specific antibodies and cellular immunity than gD2t. We previously reported an HSV-2 mutant, HSV2-gD27, in which the nectin-1-binding domain of gD2 is altered so that the virus is impaired for infecting neural, but not epithelial cells, in vitro and is impaired for infecting dorsal root ganglia in mice (K Wang, JD Kappel, C Canders, et al. J Virol. 86:12891-902, 2012. doi: 10.1128/JVI.01055-12). Here we report that the mutations in HSV2-gD27are stable when the virus was passaged in cell culture and during acute infection of mice. HSV2-gD27 was attenuated in mice when inoculated onto the cornea, intramuscularly (i.m.), intravaginally, and intracranially. Vaccination of mice i.m. with HSV2-gD27 provided better inhibition of challenge virus replication in the vagina than when the virus was used to vaccinate mice intranasally or subcutaneously. Comparison of i.m. vaccination of HSV2-gD27 versus gD2t in adjuvant showed that HSV2-gD27 induced better reduction of challenge virus replication in the vagina and reduced latent viral load in dorsal root ganglia, but induced lower serum neutralizing antibody titers than gD2t in adjuvant. Taken together, our data indicate that a live attenuated HSV2 vaccine impaired for infection of neurons provides better protection from vaginal challenge with HSV-2 than a subunit vaccine despite inducing lower titers of HSV-2 neutralizing antibodies in the serum.

    IMPORTANCE Genital herpes simplex (HSV) is one of the most prevalent sexually transmitted diseases. Though HSV-2 disease is usually mild, it can be life threatening in neonates and immunocompromised persons. In addition, genital herpes increases the frequency of HIV infection and transmission. HSV-2 maintains a latent infection in sensory neurons and cannot be cleared with antiviral drugs. The virus frequently reactivates, resulting in virus shedding in the genital area which serves as source for transmission. A prophylactic vaccine is needed to prevent disease and control the spread of the virus. Previous human trials of subunit vaccines have been unsuccessful. Here, we report the results of vaccinating mice with a new type of live attenuated HSV2 vaccine that is impaired for infection of neurons and provides better protection of mice than a subunit vaccine. The strategy of altering cell-tropism of a virus is a new approach for a live attenuated vaccine.

  • Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors [PublishAheadOfPrint]

  • Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, only retaining clinically-useful susceptibility to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38-49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PR4) and compared the phenotypic sensitivity (EC50) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PR4 showed PI susceptibility equivalent to or greater than HIV-1 for all PI. We also compared crystallographic structures of WT HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PR4 enzyme. These models suggests that the amprenavir sensitivity of PR4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2rrsquo; pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2.

    IMPORTANCE: Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids, and testing the PI susceptibility of the resulting viruses. We found that the HIV-2 clone containing all four changes (PR4) was as susceptible as HIV-1 to all nine PI. We also modeled the PR4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease may be the primary cause of HIV-2 PI resistance.

  • Viral regulation of cell tropism in human cytomegalovirus [PublishAheadOfPrint]

  • The viral glycoproteins that decorate enveloped viruses play crucial roles in cell entry and in large part dictate the spectrum of cell types that a virus can infect. The identification in human cytomegalovirus (HCMV) of a viral ER resident glycoprotein that regulates the composition of alternative viral envelope glycoprotein complexes raises the intriguing possibility that certain viruses might actively regulate the tropism of progeny virions to improve their fitness or to navigate through the host.

  • Zoonotic Potential of Simian Arteriviruses [PublishAheadOfPrint]

  • Wild non-human primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild non-human primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host-species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be "pre-emergent" zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.

  • Epstein-Barr Virus Tegument Protein BGLF2 Promotes EBV Reactivation Through Activation of the p38 Mitogen-Activated Protein Kinase [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-B promoter activity using AP-1 and NF-B luciferase reporter assays. We found that EBV BGLF2 activated AP-1, but not NF-B, reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK)- two proteins important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in EBV latently infected lymphoblastoid cell lines and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinase 1 and 2 (MEK1/2), reduced expression of BZLF1 and virus production induced by BGLF2. In summary, EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production.

    Importance Epstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression, or inhibition of p38/MAPK which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein, which is delivered to cells upon infection, activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

  • Correlation between virus replication and antibody responses in macaques following infection with pandemic influenza A virus. [PublishAheadOfPrint]

  • Influenza virus infection of non-human primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection and there is no information on relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce in humans and largely confined to evaluation of attenuated virus strains. Here we have compared the effect of a commonly used dose (4 x 106 TCID50) versus a hundred fold higher dose, given by intra-bronchial installation, in two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of IFN in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high dose group it was present at higher levels and for a longer duration. The virus specific antibody response was not significantly different between the groups. However, antibody ELISA, virus neutralization and hemaglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between level of virus production upon infection and induction of functional antibody responses against the virus.

    IMPORTANCE There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or non-human primates. We showed only a marginal and variable effect of virus dose on virus production in the trachea, but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition titer and antibody dependent cell mediated cytotoxicity, correlated with the level of virus replication measured in the trachea. The study reveals a relationship between virus production and functional antibody formation, which could be relevant in defining appropriate criteria for new influenza virus vaccine candidates.

  • Specialization of hepatitis C virus envelope glycoproteins for B-lymphocytes in chronically infected patients [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several evidences have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B-lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B-lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B-lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B-lymphocytes onto the HCV JFH-1 replicating virus switched the entry tropism of this non-lymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B-cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence and extrahepatic disorders.

    IMPORTANCE Hepatitis C Virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B-lymphocytes that conferred to a non lymphotropic HCV virus the ability to enter into B-cells, thus providing a platform for characterizing HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and non-hepatotropic HCV populations may be important for effective treatment of chronically infected patients.

  • A betabaculovirus-encoded gp64 homolog is a functional envelope fusion protein [PublishAheadOfPrint]

  • The envelope fusion protein GP64 is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog is a functional envelope fusion protein and could enable infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

  • Differential recognition of influenza A viruses by M158-66 epitope-specific CD8+ T cells is determined by extra-epitopic amino acid residues [PublishAheadOfPrint]

  • Natural influenza A virus infections elicit both virus-specific antibody and CD4+ and CD8+ T cell responses. Influenza A virus-specific CD8+ cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade from recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201 restricted matrix protein 1 (M1)58-66 epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extra-epitopic amino acid substitutions. Activation of specific T cells was used as read out for antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrated that naturally occurring variation at extra-epitopic amino acid residues affect CD8+ T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs, while retaining the conserved amino acid sequence of the epitope, which may represent a yet unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses.

    IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8+ cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes mutations occur that allow influenza A viruses to evade from recognition by CTLs. However, the immunodominant HLA-A*0201 restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights in the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

  • Structural Insights into HIV-1 Vif-APOBEC3F Interaction [PublishAheadOfPrint]

  • The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1, F2, and F3 box motifs are essential for both A3C and A3F degradation, and additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1, F2, and F3 box motifs sterically cluster to form a flexible A3F-interaction interface, which represents hydrophobic and positively-charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.

    IMPORTANCE HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F-interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than Vif-A3C's. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

  • Structural Studies of Chikungunya Virus-like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission [PublishAheadOfPrint]

  • Chikungunya is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor-binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1, and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can however enter the next cell by travelling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur.

    IMPORTANCE Alphaviral infections are mainly transmitted by mosquitoes. Chikungunya virus (CHIKV), which belongs to the alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

  • Priming of the Respiratory Tract with Immunobiotic Lactobacillus plantarum Limits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM) [PublishAheadOfPrint]

  • Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces many clinical and pathologic features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, a bacterial artificial chromosome-based recombinant PVM strain J3666 that incorporates the fluorescent tag, monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. With recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c+MHC11+) and alveolar macrophages (AMs; CD11c+SiglecF+) in vivo, and likewise detect mKATE2+ DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum (Lp), a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM, and a reduction from ~40% to fewer than 10% mKATE2+ AMs in association with 2-log drop in release of infectious virions. In contrast, AMs from Lp-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response.

    IMPORTANCE Pneumonia Virus of Mice (PVM) is a natural mouse pathogen that serves as a model for severe human respiratory syncytial virus disease. We have developed a fully-functional recombinant PVM with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide us with insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum.


  • Upon release of HIV-1 particles from the infected cell, the viral protease cleaves the Gag polyprotein at specific sites, triggering maturation. During this process, which is essential for infectivity, the capsid protein (CA) reassembles into a conical core. Maturation inhibitors (MIs) block HIV-1 maturation by interfering with protease-mediated CA-SP1 processing and by stabilizing the immature CA-SP1 lattice; virions from MI-treated cells retain an immature-like CA-SP1 lattice, whereas mutational abolition of cleavage at the CA-SP1 site results in virions in which the CA-SP1 lattice converts to a mature-like form. We previously reported that propagation of HIV-1 in the presence of MI PF-46396 selected for assembly-defective, compound-dependent mutants with amino acid substitutions in the major homology region (MHR) of CA. Propagation of these mutants in the absence of PF-46396 resulted in the acquisition of second-site compensatory mutations. These included a Thr-to-Ile substitution at SP1 residue 8 (T8I), which results in impaired CA-SP1 processing. Thus, the T8I mutation phenocopies PF-46396 treatment in terms of its ability to rescue the replication defect imposed by the MHR mutations, and to impede CA-SP1 processing. Here, we use cryo-electron tomography to show that, like MIs, the T8I mutation stabilizes the immature-like CA-SP1 lattice. These results have important implications for the mechanism of action of HIV-1 MIs; they also suggest that T8I may provide a valuable tool for structural definition of the CA-SP1 boundary region, which has thus far been refractory to high-resolution analysis apparently because of conformational flexibility in this region of Gag.

    IMPORTANCE HIV-1 maturation involves dissection of the Gag polyprotein by the viral protease, and assembly of a conical capsid enclosing the viral ribonucleoprotein. Maturation inhibitors (MIs) prevent the final cleavage step at the site between the capsid protein (CA) and the spacer peptide 1 (SP1), apparently by binding at this site and denying the protease access. Additionally, MIs stabilize the immature-like CA-SP1 lattice, preventing release of CA into the soluble pool. We previously found that a mutation in SP1, T8I, rescues a PF-46396-dependent CA mutant and blocks CA-SP1 cleavage. In this study, we imaged T8I virions by cryo-electron tomography and show that T8I mutants, like MI-treated virions, contain an immature CA-SP1 lattice. These results lay the groundwork needed to understand the structure of the CA-SP1 interface region and further illuminate the mechanism of action of MIs.

  • Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62 [PublishAheadOfPrint]

  • The immediate-early 62 protein (IE62) of varicella-zoster virus (VZV), a major viral trans-activator, initiates the virus life cycle and is a key component of pathogenesis. The IE62 possesses several domains essential for trans-activation, including an acidic trans-activation domain (TAD), a serine-rich tract (SRT), and binding domains for USF, TFIIB, and TATA-box binding protein (TBP). Transient transfection assays showed that the VZV IE62 lacking the SRT trans-activated the early VZV ORF61 promoter by only 16% of that by the full-length IE62. When the SRT of IE62 was replaced with the SRT of equine herpesvirus 1 (EHV-1) IEP, its trans-activation activity was completely restored. Herpes simplex virus type 1 (HSV-1) ICP4 that lacks a TAD very weakly (1.5-fold) trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein exhibited trans-activation ability (10.2-fold), indicating that the IE62 TAD functions with the SRT of HSV-1 ICP4 to trans-activate viral promoters. When the serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly, trans-activation activities of the modified IE62 proteins were reduced to 39% and 18% of wild-type activity, respectively. Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of IE62 interacted with nucleolar-ribosomal protein EAP, which resulted in the formation of globular structures within the nucleus. These results suggest that the SRT plays an important role in VZV viral gene expression and replication.

    IMPORTANCE The immediate-early 62 protein (IE62) of varicella-zoster virus (VZV) is a major viral trans-activator and is essential for viral growth. Our data show that the serine-rich tract (SRT) of VZV IE62, which is well conserved within the alphaherpesviruses, is needed for trans-activation mediated by the acidic trans-activation domain (TAD). The TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with cellular Mediator 25 in bimolecular complementation assays. The interaction of the IE62 SRT with nucleolar-ribosomal protein EAP resulted in the formation of globular structures within the nucleus. Understanding the mechanisms by which the TAD and SRT of IE62 contribute to the function of this essential regulatory protein is important in understanding the gene program of this human pathogen.

  • Induction of lytic cycle sensitizes Epstein-Barr virus infected B cells to NK cell killing that is counteracted by virus-mediated NK cell evasion mechanisms in late lytic cycle [PublishAheadOfPrint]

  • Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV infected cells undergoing lytic replication in-vitro and studies in both humans, and mice with reconstituted human immune systems have shown NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV infected cells undergoing lytic replication, and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as yet unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, whilst cells entering lytic cycle become sensitized to NK cell killing, we observed that cells in late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses.

    IMPORTANCE This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV infected B cells undergoing lytic replication is dependent upon the phase of lytic cycle. Induction of lytic cycle is associated with acquired sensitization to NK cell killing, while progress through late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, implicating important roles for the BZLF1 immediate-early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.

  • Epstein-Barr Virus BZLF1-Mediated Down-regulation of Pro-inflammatory Factors is Essential for Optimal Lytic Viral Replication [PublishAheadOfPrint]

  • Elevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, the inflammatory response and its biological significance during the lytic EBV cycle remain elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the pro-inflammatory factor TNFaalpha; by binding to the promoter of TNFaalpha; and preventing NFB activation. A BZLF1207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the pro-inflammatory factors TNFaalpha; and IFN and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNFaalpha; depletion restored the viral replication mediated by BZLF1207-210. Furthermore, a combination of TNFaalpha;- and IFN-neutralizing antibodies recovered BZLF1207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNFaalpha; and IFN secretion during the lytic cycle. These results suggest that the EBV BZLF1 attenuates the pro-inflammatory responses to facilitate viral replication.

    IMPORTANCE The pro-inflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as transcriptional suppressor of the inflammatory factors TNFaalpha; and IFN and confirm that BZLF1-facilitated escape from the TNFaalpha; and IFN response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to evade the antiviral inflammatory response.

  • A subset of CD4/CD8 double negative T cells expresses HIV proteins in patients on ART [PublishAheadOfPrint]

  • A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of anti-retroviral therapy (ART), which reseed viremia after treatment is stopped. In general it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view, and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4- cells. Here, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and Fiber-Optic Array Scanning Technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef and Env-mediated receptor internalization. We also extend our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double negative aalpha;/bbeta; T cells that express HIV protein may be a component of the long-lived reservoir.

    IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during anti-retroviral therapy (ART) that leads to rebound of virus if treatment is stopped. Here, we use flow cytometry and cell imaging to characterize protein expression in HIV infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins such as Env and Nef. Gag+CD4- cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

  • KAP1 is a host restriction factor that promotes HAdV E1B-55K SUMO modification [PublishAheadOfPrint]

  • Once transported to the replication sites, HAdVs need to assure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly characterized, but represent a decisive moment in establishing a productive infection. Here, we identify a novel host viral restriction factor, KAP1. This heterochromatin associated transcription factor regulates the dynamic organization of host chromatin structure via its ability to influence epigenetic marks and chromatin compaction. In response to DNA damage, KAP1 is phosphorylated and functionally inactive, resulting in chromatin relaxation. We discovered that KAP1 posttranslational modification is dramatically altered during HAdV infection to limit the antiviral capacity of this host restriction factor, which represents an essential step required for efficient viral replication. Conversely, we also observed an HAdV-mediated decrease of KAP1 SUMO moieties during infection, known to promote chromatin-decondensation events. Based on our findings, we provide evidence that HAdV induces KAP1 deSUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism.

    IMPORTANCE Here we describe a novel cellular restriction factor for Human Adenovirus (HAdV) that sheds light on very early modulation processes in viral infection. We reported that chromatin formation and cellular SWI/SNF chromatin remodeling play a key role in HAdV transcriptional regulation (1-4). We observed that the cellular chromatin-associated factor, and epigenetic reader SPOC1 represses HAdV infection and gene expression. Here, we illustrate the role of the SPOC1 interacting factor KAP1 during productive HAdV growth. KAP1 binds to the viral E1B-55K protein, promoting its SUMO modification, therefore illustrating a crucial step for efficient viral replication. Simultaneously, KAP1 posttranslational modification is dramatically altered during infection. We observed an HAdV-mediated decrease in KAP1 SUMOylation, known to promote chromatin-decondensation events. These findings indicate that HAdV induces loss of KAP1 SUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism.

  • Small interfering RNA pathway modulates initial viral infection in the midgut epithelium of insect after ingestion of virus [PublishAheadOfPrint]

  • Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. A rice virus (southern rice black streaked dwarf virus, SRBSDV) is restricted its infection in the midgut epithelium of its incompetent vector (small brown planthopper, SBPH). Here, we showed that the siRNA pathway was triggered by SRBSDV infection in the continuous cultured cells derived from SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of siRNA pathway due to RNA interference strongly increased the propagation ability of SRBSDV in the continuous SBPH cultured cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach a certain threshold (1.99 x 109 copies of SRBSDV P10 gene/mmu;g midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. The silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results revealed a new finding that the siRNA pathway can control the initial infection of a virus in the insect midgut epithelium, which finally affected vector competence of a virus.

    IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck for viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is exclusively restricted to the midgut epithelium of the incompetent vector small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of siRNA pathway increased viral titers in the midgut epithelium past a certain threshold (1.99 x 109 copies of SRBSDV P10 gene/mmu;g midgut RNA) for viral dissemination into the midgut muscles, then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence of a direct role of the siRNA antiviral pathway in controlling viral dissemination from the midgut epithelium and affecting vector competence of a virus.

  • Vpr-dependent and RT-dependent components of HIV-1-induced cell death in resting peripheral blood CD4 T cells, and rescue by common gamma chain cytokines [PublishAheadOfPrint]

  • HIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms. In vivo, HIV-1 infects both activated and resting CD4 T cells, but in vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription, resulting in abortive infection. Here, we show that in vitro HIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection. In vivo, however, various micro-environmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma chain cytokines (CGCC) such as IL-7 render resting CD4 T cell permissible to HIV-1 infection without inducing T cell activation. Here we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidence by early release of cytochrome C and caspase 3/7 activation. Cell death is triggered by both products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1 induced cell death in this system. These results show that a major restriction to HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection.

    IMPORTANCE A major consequence of HIV-1 infection is the destruction of CD4 T cells. Here we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in various cell types, we show for the first time their concerted effect in inducing resting CD4 T cell depletion. Importantly, we found that cytokines such as IL-7 or IL-4, which are particularly active in sites of HIV-1 replication, protect resting CD4 T cells from these cytopathic effects, and primarily through this protection, rather than through enhancement of specific replicative steps, they promote productive infection. This study provides important new insights for the understanding of the early steps of HIV-1 infection and T cell depletion.

  • Genome-wide mapping of the binding sites and structural analysis of KSHV vIRF2 reveal it is a DNA-binding transcription factor [PublishAheadOfPrint]

  • Oncogenic herpesvirus KSHV is known to encode four viral interferon regulatory factors (vIRF1-4) to subvert host antiviral immune response, but their detailed DNA-binding profiles as transcription factors in host remain uncharacterized. Here we first performed the genome-wide vIRF2-binding sites mapping in human genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). vIRF2 was capable of binding to the promoter regions of 100 putative target genes. Importantly, we confirmed vIRF2 can specifically interact with the promoters of the genes encoding PIK3C3, HMGCR and HMGCL that are associated with autophagosomes formation or tumor progression and metastasis, and regulate their transcriptions in vivo. The crystal structure of vIRF2 DNA-binding domain (DBD) showed variable loop conformations and different positive charge distribution from vIRF1 and cellular IRFs that are associated with DNA-binding specificities. Structure-based mutagenesis revealed Arg82 and Arg85 are required for in vitro DNA binding activity of vIRF2DBD, and can abolish the transcription regulation function of vIRF2 on the promoter reporter activity of PIK3C3, HMGCR and HMGCL. Collectively, our study provided unique insights into the DNA binding potency of vIRF2 and suggested that vIRF2 could act as a transcription factor of its target genes in host antiviral immune response.

    IMPORTANCE Oncogenic herpesvirus KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castelman's disease. KSHV has developed a unique mechanism to subvert host antiviral immune responses by encoding four homologues of cellular interferon regulatory factors (vIRF1~4). However, none of their DNA-binding profiles in human genome have been characterized till now, and the structural basis for their diverse DNA-binding properties remain poorly understood. In this study, we performed the first human genome and found it can bind to the promoter regions of 100 target cellular genes. X-ray structure analysis and functional studies provided unique insights into its DNA-binding potency and regulation of target genes expression. Our study suggested that vIRF2 could act as a transcription factor of its target genes and contribute to KSHV infection and pathogenesis through versatile functions.

  • Single mutations in the VP2 300 loop region of the three-fold spike of the carnivore parvovirus capsid can determine host range [PublishAheadOfPrint]

  • Sylvatic carnivores, such as raccoons, have recently been recognized as important hosts in the evolution of canine parvovirus (CPV), a pandemic pathogen of domestic dogs. Although viruses from raccoons do not efficiently bind the dog transferrin receptor (TfR) or infect dog cells, a single mutation changing an aspartic acid to a glycine at capsid (VP2) position 300 in the prototype raccoon CPV allows dog cell infection. As VP2 position 300 exhibits extensive amino acid variation among the carnivore parvoviruses, we further investigated its role in determining host range by analyzing its diversity and evolution in nature, and by creating a comprehensive set of VP2 position 300 residues in infectious clones. Notably, some position 300 residues rendered CPV non-infectious for dog, but not cat or fox, cells. Changes of adjacent residues (299 and 301) were also often observed after cell culture passage in different hosts, and some of those mimicked changes seen in viruses recovered from natural infections of alternative hosts, suggesting that compensatory mutations were selected to accommodate the new 300 residue. Analysis of the TfRs of carnivore hosts used in the experimental evolution studies demonstrated that their glycosylation patterns varied, including a glycan present only on the domestic dog TfR that dictates parvovirus susceptibility. Overall, there were significant differences in the ability of viruses with alternative 300 residues to bind TfRs and infect different carnivore hosts, demonstrating that the process of infection is highly host-dependent and that VP2 position 300 is a key determinant of host range.

    IMPORTANCE Although the emergence and pandemic spread of canine parvovirus (CPV) has been well documented, the carnivore hosts and evolutionary pathways involved in its emergence remain enigmatic. We recently demonstrated that a region in the capsid structure of CPV, centered around VP2 position 300, varies after transfer to alternative carnivore hosts and may allow infection of previously non-susceptible hosts in vitro. Here, we show that VP2 position 300 is the most variable residue in the parvovirus capsid in nature, suggesting it is a critical determinant in the cross-species transfer of viruses between different carnivores due to its interactions with the transferrin receptor to mediate infection. To this end, we demonstrated that there are substantial differences in receptor binding and infectivity of various VP2 position 300 mutants for different carnivore species, and that single mutations in this region can influence whether a host is susceptible or refractory to virus infection.

  • Extracellular vesicles mediate receptor-independent transmission of novel tick-borne bunyavirus [PublishAheadOfPrint]

  • Severe fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genus Phlebovirus in the family Bunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12-30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the IFN-bbeta; promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens.

    IMPORTANCE SFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We asked if these "infectious" extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point towards a novel mechanism for virus replication among arthropod-borne viruses.

  • Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles [PublishAheadOfPrint]

  • Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children that can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues for VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine change were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to EV71 entry inhibitor suramin which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles.

    IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are currently available for the prevention or treatment of EV71 infection. Understanding the determinants of virion assembly and entry will facilitate vaccine development and drug discovery. Here, we identified twenty three out of twenty seven positively charged residues in VP1, which impaired or blocked the production of infectious particles. The defect could be rescued by second-site mutations within the VP1 protein. Our findings highlight the importance of positively charged residues in VP1 during infectious particles production and reveal a potential strategy for blocking EV71 infections by inhibiting intra- or intermolecular interactions of the VP1 protein.

  • HIV-1 Adapts to Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2 [PublishAheadOfPrint]

  • Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional post-entry blocks in common marmoset peripheral blood mononuclear cells. Here we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase the viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors.

    IMPORTANCE HIV-1 can only infect humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study we have observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and characterize the mechanisms of escape. These studies can help in the development of a novel animal model for in vivo infection of marmosets with HIV-1-like viruses.

  • VP24 is a chitin-binding protein involved in white spot syndrome virus infection [PublishAheadOfPrint]

  • Oral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186-200 in the C-terminus of VP24 were required for chitin binding. Moreover, the P-VP24186-200 peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186-200 treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection and provide new ideas for preventing WSSV infection in shrimp farms.

    IMPORTANCE In this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186-200 in the C-terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms.

  • The conserved role of an N-linked glycan on the surface antigen of HIV-1 modulating virus sensitivity to broadly neutralizing antibodies against the receptor and coreceptor binding sites [PublishAheadOfPrint]

  • HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies as compared to its WT counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 representing different clades, tissue origins, coreceptor usage, and neutralization sensitivity. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop- directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability or accessibility of bNAb epitopes in the CD4bs and coreceptor-binding region, thus representing a potential target for the design of immunogens and therapeutics.

    IMPORTANCE N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env to mediate viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor- and coreceptor-binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.

  • Structure and function of the N-terminal domain of the vesicular stomatitis virus RNA polymerase [PublishAheadOfPrint]

  • Viruses have varied mechanisms to duplicate their genomes and produce viral specific mRNAs. Negative strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from the Rhabdoviridae, Paramyxoviridae and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8AAring; resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, included segmented negative-strand RNA and dsRNA viruses.

    IMPORTANCE Negative strand RNA viruses include a diverse set of viral families that infect animals and plants causing serious illness and economic impact. This group of viruses share a common set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genomic- and subgenomic-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative strand RNA viruses.

  • Prion-type dependent deposition of PRNP-allelic products in heterozygous sheep. [PublishAheadOfPrint]

  • Susceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild type homozygous ARQ/ARQ sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In BSE infection the polymorphism effect is quite different, though the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease resistant prion protein (PrPres) from brain of heterozygous ARR/VRQ scrapie infected sheep was compared with that of BSE infected sheep with similar genotype. The triplex-Western blotting technique was used to estimate the two allotype PrP fractions in PrPres material from BSE infected ARR/VRQ sheep. PrPres in BSE contained equimolar amounts of VRQ- and ARR-PrP which contrasts with the excess (ggt;95%) VRQ-PrP fraction found in scrapie. This is evidence that TSE agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state) determine the polymorphic dependence of the PrPSc accumulation process in prion formation as well as the disease associated phenotypic expressions in the host.

    IMPORTANCE Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts as known for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie infected sheep that produce two PrP variants associated with opposite scrapie susceptibility (136V-PrP, high; 171R-PrP, very low) contain in their prion material over 95% of the 136V PrP variant. However, when infected with prions from cattle (BSE), both PrP variants occur in equal ratios. This shows that the infecting prion-type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

  • Fluid Spatial Dynamics of West Nile Virus in the USA: Rapid Spread in a Permissive Host Environment [PublishAheadOfPrint]

  • The introduction of West Nile virus (WNV) into North America in 1999 is a classical example of viral emergence in a new environment, with its subsequent dispersion across the continent having a major impact on local bird populations. Despite the importance of this epizootic, the pattern, dynamics and determinants of WNV spread in its natural hosts remain uncertain. In particular, it is unclear whether the virus encountered major barriers to transmission, or spread in an unconstrained manner, and if specific viral lineages were favored over others indicative of intrinsic differences in fitness. To address these key questions in WNV evolution and ecology we sequenced the complete genomes of approximately 300 avian isolates sampled across the USA between 2001-2012. Phylogenetic analysis revealed a relatively llsquo;star-like' tree structure, indicative of explosive viral spread in US, although with some replacement of viral genotypes through time. These data are striking in that viral sequences exhibit relatively limited clustering according to geographic region, particularly for those viruses sampled from birds, and no strong phylogenetic association with well sampled avian species. The genome sequence data analysed here also contain relatively little evidence for adaptive evolution, particularly on structural proteins, suggesting that most viral lineages are of similar fitness, and that WNV is well adapted to the ecology of mosquito vectors and diverse avian hosts in the USA. In sum, the molecular evolution of WNV in North America depicts a largely unfettered expansion within a permissive host and geographic population with little evidence of major adaptive barriers.

    IMPORTANCE How viruses spread in new host and geographic environments is central to understanding the emergence and evolution of novel infectious diseases, and for predicting their likely impact. The emergence of the vector-borne West Nile virus (WNV) in North America in 1999 represents a classic example of this process. Using approximately 300 new viral genomes sampled from wild birds we show that WNV experienced an explosive spread with little geographical or host constraints within birds, and relatively low levels of adaptive evolution. From its introduction into New York State, WNV spread across the USA, reaching California and Florida within four years, a migration that is clearly reflected in our genomic sequence data, and with a general absence of distinct geographical clusters of bird viruses. However, some geographically distinct viral lineages were found to circulate in mosquitoes, likely reflecting their limited long distance movement compared to avian species.

  • Hemagglutinin stalk- and neuraminidase-specific monoclonal antibodies protect against lethal H10N8 influenza virus infection in mice [PublishAheadOfPrint]

  • Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed mAbs and further characterized the binding reactivity of these antibodies (via ELISA) to a range of purified virus and recombinant protein substrates. The H10-directed mAbs displayed functional hemagglutination inhibition (HI) and neutralization activity and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies as well as a previously generated, group 2 HA stalk-reactive antibody demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the hemagglutinin may be efficacious in preventing influenza virus-induced morbidity and mortality.

    IMPORTANCE Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. While these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and re-assortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. Here we generated a panel of H10- and N8-reactive mAbs. While these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of mAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.

  • HSV-1 infection of tree shrews differs from that of mice in the severity of acute infection and viral transcription in the peripheral nervous system [PublishAheadOfPrint]

  • Studies of HSV infections of humans are limited by the use of rodent models such as mice, rabbits and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic and evolutional levels, tree shrews are much closer to primates than rodents, and tree shrews are susceptible to HSV infection. Thus we have studied HSV-1 infection in the tree shrew trigeminal ganglion following ocular inoculation. In situ hybridization, PCR and qRT-PCR analyses confirm that HSV-1 latently infects neurons of the trigeminal ganglion (TG). When explant co-cultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of co-cultivation with high efficiency. Swabbing of the cornea of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes including ICP0, ICP4 and LAT. In acutely infected tree shrew TGs, no detectable level of ICP4 was seen, suggesting the absence of, or a very weak, acute infection compared to the mouse. Immunofluorescent staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high level of ICP0 transcript in addition to LAT during latency. However the ICP0 transcript remained nuclear and no ICP0 protein could be seen in during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models.

    IMPORTANCE Herpes Simplex Viruses (HSV) establish life long infection in more than 80% of the population and their reactivation leads to oral and genital Herpes. Currently rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. At behavioral, anatomical, genomic and evolutional levels, tree shrews are much closer to primates than rodents. In an attempt to further develop the tree shrew as a useful model to study herpes virus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant co-cultivation and that spontaneous reactivation could be detected by cell culture of eye tears. Interestingly, the tree shrew model is quite different from the mouse model of HSV infection, in that the virus exhibited only a mild acute infection following inoculation with no detectable infectious virus from the sensory neurons. The mild infection may be more similar to human infection in that the sensory neurons continue to function after Herpes reactivation and the affected skin tissue does not lose sensation. Our findings suggest that the tree shrew is a viable model to study HSV latency.

  • Knockout of the host resistance gene Pkr fully restores replication of murine cytomegalovirus (MCMV) m142 and m143 mutants in vivo [PublishAheadOfPrint]

  • MCMV proteins m142 and m143 are essential for viral replication. They bind double-stranded RNA and prevent protein kinase R-induced protein synthesis shutoff. Whether the two viral proteins have additional functions as their homologs in human cytomegalovirus do, remained unknown. We show that MCMV m142 and m143 knockout mutants attain organ titers equivalent to wildtype MCMV in Pkr knockout mice, suggesting that these viral proteins do not encode additional PKR-independent functions relevant for pathogenesis in vivo.

  • Chemical Cross-linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens [PublishAheadOfPrint]

  • Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env, but nevertheless sample different conformational states. Here, we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or non-neutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151 and 3BC315, and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins, and by extrapolation also vaccine immunogens from other pathogens.

    IMPORTANCE The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However these trimers are conformationally flexible, potentially reducing B cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of non-neutralizing antibodies whilst largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody-affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.

  • Modulation of the Mitochondrial Antiviral Signaling by Human Herpesvirus 8 Interferon Regulatory Factor 1 [PublishAheadOfPrint]

  • Mitochondrial lipid raft-like microdomains, experimentally also termed mitochondrial detergent resistant membrane fractions (mDRM), play a role as platforms for recruiting signaling molecules involved in antiviral responses such as apoptosis and innate immunity. Viruses can modulate mitochondrial functions for their own survival and replication. However, viral regulation of the antiviral responses via mDRM remains incompletely understood. Here, we report that human herpesvirus 8 (HHV-8) gene product viral interferon regulatory factor 1 (vIRF-1) is targeted to mDRM during virus replication and negatively regulates the mitochondrial antiviral signaling (MAVS) protein-mediated antiviral responses. The N-terminal region of vIRF-1 interacts directly with membrane lipids including cardiolipin. In addition, a GxRP motif within the N-terminus of vIRF-1, conserved in the mDRM-targeting region of mitochondrial proteins including PINK1 and MAVS, was found to be important for vIRF-1 association with mitochondria. Furthermore, MAVS, which has the potential to promote vIRF-1 targeting to mDRM potentially by inducing cardiolipin exposure on the outer membrane of mitochondria, interacts with vIRF-1, which in turn inhibits MAVS-mediated antiviral signaling. Consistent with these results, vIRF-1 targeting to mDRM contributes to promotion of HHV-8 productive replication and inhibition of associated apoptosis. Combined, our results suggest novel molecular mechanisms for negative feedback regulation of MAVS by vIRF-1 during virus replication.

    IMPORTANCE Successful virus replication is in large part achieved by the ability of viruses to counteract apoptosis and innate immune responses elicited by infection of host cells. Recently, mitochondria have emerged to play a central role in antiviral signaling. In particular, mitochondrial lipid raft-like microdomains appear to function as platforms in cell apoptosis signaling. However, viral regulation of antiviral signaling through the mitochondrial microdomains remains incompletely understood. The present study demonstrates that HHV-8-encoded vIRF-1 targets to the mitochondrial detergent resistant microdomains via direct interaction with cardiolipin and inhibits MAVS protein-mediated apoptosis and type I interferon gene expression in a negative feedback manner, thus promoting HHV-8 productive replication. These results suggest that vIRF-1 is the first example of a viral protein to inhibit mitochondrial antiviral signaling through lipid raft-like microdomains.

  • Dengue virus prM-specific human monoclonal antibodies with virus replication enhancing properties recognize a single immunodominant antigenic site [PublishAheadOfPrint]

  • The proposed antibody dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the pre-membrane (prM) protein. Here we report the isolation of a large panel of naturally-occurring human mAbs obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural dengue virus infections or following primary DENV serotype 1 live attenuated virus vaccination, to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human mAbs, encoded by diverse antibody variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of varying clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing DENV infection of Fc receptor-bearing cells.

    IMPORTANCE Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals following infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.

  • A molecular sensor to characterize arenavirus envelope glycoprotein cleavage by subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) [PublishAheadOfPrint]

  • Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses only genetic information is available and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears therefore as a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate efficiency and subcellular location of arenavirus GPC processing. In proof-of-concept, our sensor correctly predicts efficient processing of the GPC of the newly emerged pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and non-pathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system to assess processing of putative cleavage sites derived from newly discovered arenavirus GPC by SKI-1/S1P of humans or any other species, based solely on sequence information.

    IMPORTANCE Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive infection of arenaviruses in human cells is processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). In order to break the species barrier during zoonotic transmission and to cause severe disease in man, newly emerging arenaviruses must be able to efficiently hijack human SKI-1/S1P. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emerged pathogenic Lujo virus by human SKI-1/S1P. Our sensor represents thus a rapid and robust test system to assess if the glycoprotein of any newly emerging arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information.


  • HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved llsquo;late domain' motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6Gag), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gag significantly increased virus infectivity and replication capacity compared to viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gag confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy.

    IMPORTANCE Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease-inhibitor (PI) therapy. However, the reason these duplications arise, and their consequences for virus replication, remain to be established. In this study, we examine the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI-resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI-resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

  • The intricate roles of mammalian sirtuins in defense against viral pathogens [PublishAheadOfPrint]

  • For a number of years, sirtuin enzymes have been appreciated as effective "sensors" of the cellular environment to rapidly transmit information to diverse cellular pathways. Much effort was placed into exploring their roles in human cancers and aging. However, a growing body of literature brings these enzymes to the spotlight in the field of virology. Here, we discuss sirtuin functions in the context of viral infection, which provide regulatory points for therapeutic intervention against pathogens.

  • A naturally occurring domestic cat APOBEC3 variant confers resistance to FIV infection [PublishAheadOfPrint]

  • Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated anti-viral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I-VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has yet to be addressed. In this study, we demonstrate that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also reveal that the codon 65 of feline A3Z3 is a positively selected site, and that A3Z3 hap V is under positive selection during evolution. Particularly noteworthy, feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not its hydrophobicity, of the amino acid positioned at 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses infer that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected to escape from an ancestral FIV. This is the first evidence for an evolutionary arms race between the domestic cat and its cognate lentivirus.

    IMPORTANCE Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., HIV-1 and SIV) if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat that was likely influenced by lentiviral infection.

  • Conditional immune escape during chronic SIV infection [PublishAheadOfPrint]

  • Anti-HIV CD8 T cells included in therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Identifying which epitopes do not accumulate variants, but retain immunogenicity, depends on both host MHC genetics and the likelihood for an epitope to tolerate variation. We previously found that immune escape during acute SIV infection is conditional; the accumulation of mutations in T cell epitopes is limited and the rate of accumulation depends on the number of epitopes being targeted. We now test the hypothesis that conditional immune escape extends into chronic SIV infection and that epitopes with preserved wild-type sequence have the potential to elicit epitope-specific CD8 T cells. We deep sequenced SIV from Mauritian cynomolgus macaques (MCMs) that were homozygous and heterozygous for the M3 MHC haplotype and had been infected with SIV for about one year. When interrogating variation within individual epitopes restricted by M3 MHC alleles, we found three categories of epitopes, that we called Categories A, B, and C. Category B epitopes readily accumulated variants in M3 homozygous MCMs, but this was less common in M3 heterozygous MCMs. We then determined that chronic CD8 T cells specific for these epitopes were more likely preserved in the M3 heterozygous MCMs, when compared to M3 homozygous MCMs. We provide evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are homozygous for a set of MHC alleles are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles.

    IMPORTANCE Anti-HIV CD8 T cells that are part of therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Defining these epitope sequences is a necessary precursor to designing approaches that enhance the functionality of CD8 T cells with the potential to control virus replication during chronic infection or after reactivation of latent virus. Using MHC homozygous and heterozygous Mauritian cynomolgus macaques, we now provide evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are MHC homozygous are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. Importantly, our findings support the conditional immune escape hypothesis, such that the potential to present a greater number of CD8 T cell epitopes within a single animal can delay immune escape in targeted epitopes. As a result, certain epitope sequences can retain immunogenicity into chronic infection.

  • Defensive perimeter in the central nervous system: predominance of infected astrocytes and astrogliosis during recovery from varicella-zoster virus encephalitis [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types (neurons, microglia, oligodendrocytes, astrocytes). The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytia formation within the aggregates included the continuity of cytoplasm positive for the VZV gH fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis.

    Importance. Investigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples under which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons.

  • Characterization of a chimera herpes simplex virus 1 (HSV-1) in which its Us3 protein kinase gene was replaced with the HSV-2 Us3 gene [PublishAheadOfPrint]

  • Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) play important roles in viral replication and pathogenicity. To investigate type-specific differences between HSV-1 Us3 and HSV-2 Us3 in cells infected by viruses with all the same viral gene products except for their Us3 kinase, we constructed and characterized a recombinant HSV-1 in which its Us3 gene was replaced with the HSV-2 Us3 gene. Replacement of HSV-1 Us3 with HSV-2 Us3 had no apparent effect on viral growth in cell cultures or on the range of proteins phosphorylated by Us3. HSV-2 Us3 efficiently compensated for HSV-1 Us3 functions, including blocking apoptosis, controlling infected cell morphology, and down-regulating cell surface expression of viral envelope glycoprotein B. In contrast, replacement of HSV-1 Us3 by HSV-2 Us3 changed the phosphorylation status of UL31 and UL34, which are critical viral regulators of nuclear egress. It also caused aberrant localization of these viral proteins, aberrant accumulation of primary enveloped virions in membranous vesicle structures adjacent to the nuclear membrane, and reduced viral cell-cell spread in cell cultures and pathogenesis in mice. These results clearly demonstrated biological differences between HSV-1 Us3 and HSV-2 Us3, especially in regulation of viral nuclear egress and phosphorylation of viral regulators critical for this process. Our study also suggested that the regulatory role(s) of HSV-1 Us3, which were not carried out by HSV-2 Us3 was important for HSV-1 cell-cell spread and pathogenesis in vivo.

    IMPORTANCE A previous study comparing the phenotypes of HSV-1 and HSV-2 suggested that the HSV-2 Us3 kinase lacked some of the functions of HSV-1 Us3 kinase. The difference between HSV-1 and HSV-2 Us3 kinases appeared to be because some Us3 phosphorylation sites in HSV-1 proteins are not conserved in the corresponding HSV-2 proteins. Therefore, we generated recombinant HSV-1 viruses YK781 (Us3-chimera) with HSV-2 Us3 and its repaired virus YK783 (Us3-repair) with HSV-1 Us3, to compare the activities of HSV-1 Us3 and HSV-2 Us3 in cells infected by viruses with the same HSV-1 gene products except for their Us3 kinase. We report here that some processes in viral nuclear egress and pathogenesis in vivo that have been attributed to HSV-1 Us3 could not be carried out by HSV-2 Us3. Therefore, our study clarified the biological differences between HSV-1 Us3 and HSV-2 Us3, which may be relevant to viral pathogenesis in vivo.

  • Env-specific IgA from Viremic HIV-infected Subjects Compromises Antibody-Dependent Cellular Cytotoxicity [PublishAheadOfPrint]

  • Elucidating the factors that modulate HIV-specific antibody-dependent cellular cytotoxicity (ADCC) will help to understand its role in HIV immunity. The aim of this study was to determine whether IgA could modify ADCC magnitude in HIV infection, abrogating its protective role. Plasma from 20 HIV+ subjects enrolled during primary HIV infection (PHI), 10 chronically infected subjects (Chronics) and 7 Elite Controllers (EC) was used. ADCC was determined using a fluorometric ADCC assay, before and after plasma IgA removal. Data was analyzed using non-parametric statistics. ADCC was documented in 80% of PHI enrollment samples and in 100% of PHI 12-month samples, Chronics and ECs; it peaked after acute infection, reached a plateau in chronic infection and decreased after ART initiation. Significant associations between ADCC and disease progression were only found after IgA plasma removal from 12-month PHI samples: ADCC magnitude not only increased after IgA removal but also correlated with CD4+T-cell preservation. This work provides evidence that gp120-specific IgA was capable of modifying ADCC responses during natural HIV infection for the first time and adds to similar evidence provided in other settings. Furthermore, it underscores the complexity of ADCC phenomenon and will help to understand its underlying mechanisms.

    Importance Although the induction of ADCC-mediating antibodies in HIV-infected subjects has been extensively documented, the association of these antibodies with protection from disease progression is poorly understood. Here, we demonstrate that plasma IgA is a factor capable of modifying the magnitude of IgG-mediated ADCC in HIV infection, mitigating its beneficial effect. These results help understand why previous studies failed to demonstrate correlations between ADCC and disease progression, and it also contribute to the notion that an HIV vaccine should stimulate the production of ADCC-mediating IgG antibodies but not IgA.

  • RNA silencing may play a role but is not the only determinant of the multiplicity of infection [PublishAheadOfPrint]

  • The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if MOI is controlled by virus functions, we have estimated MOI in plants co-infected by two genetic variants of Tomato bushy stunt virus (TBSV), by TBSV and a TBSV-derived DI-RNA, or by TBSV and a second tombusvirus, Cymbidium ringspot virus (CymRSV). MOI was significantly larger in TBSV/CymRSV co-infections ( 4.0) than in TBSV/TBSV or TBSV/DI-RNA co-infections (1.7-2.2). Co-infections of CymRSV or TBSV with chimeras in which the ORFs of one virus species was substituted by that of the other, identified a role of viral proteins in determining MOI, which ranged 1.6-3.9 according to the co-infecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Co-infections of CymRSV and TBSV mutants in which the expression of the gene silencing-suppressor protein p19 was abolished, also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that MOI is a quantitative trait showing continuous variation, and that as such it has a complex determination involving different virus-encoded functions.

    IMPORTANCE The number of viral genomes infecting a cell, or multiplicity of infection (MOI) is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, MOI may have an optimum value maximizing virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants co-infected by two genotypes of Tomato bushy stunt virus (TBSV) MOI was lower than in plants co-infected by TBSV and Cymbidium ringspot virus (CymRSV). Co-infections of CymRSV or TBSV with TBSV/CymRSV chimeras showed a role of viral proteins in MOI determination. Co-infections of CymRSV and TBSV mutants not expressing the gene silencing-suppressor protein also showed a role of gene silencing in MOI determination. Results demonstrate that MOI is a quantitative trait with a complex determination involving different viral functions.

  • Development and characterization of a guinea pig-adapted Sudan virus [PublishAheadOfPrint]

  • Infections with Sudan virus (SUDV), a member of the genus Ebolavirus, result in a severe hemorrhagic fever with a fatal outcome in over 50% of human cases. The paucity of prophylactics and therapeutics against SUDV is attributed to the lack of a small animal model to screen promising compounds. By repeatedly passaging SUDV within the livers and spleens of guinea pigs in vivo, a SUDV variant uniformly lethal to these animals (SUDV-GA) was developed, with an LD50 of 5.3 x 10-2 TCID50. Animals infected with SUDV-GA developed high viremia and died between 9 and 14 days post-infection. Several hallmarks of SUDV infection, including lymphadenopathy, increased liver enzyme activities and coagulation abnormalities were observed. Virological analyses, gross pathology, histopathology and immunohistochemistry findings indicate that SUDV-GA replicates in the livers and spleens of infected animals, similar to SUDV infections in nonhuman primates. These developments will accelerate the development of specific medical countermeasures in preparation of a future disease outbreak due to SUDV.

    Importance A disease outbreak due to Ebola virus (EBOV), suspected to have emerged during December 2013 in Guinea, is finally winding down with over 11,000 dead and 28,000 infected. Experimental EBOV vaccines and treatments were administered to patients under compassionate circumstances with promising results, and an approved countermeasure appears to be close. However, the same range of experimental candidates is not readily available against a potential disease outbreak due to other members of the genus Ebolavirus, such as Sudan virus (SUDV). One bottleneck contributing to this situation is the lack of a small animal model to screen promising drugs in an efficient and economical manner. To address this, we have generated a SUDV variant (SUDV-GA) that is uniformly lethal to guinea pigs. Animals infected with SUDV-GA develop disease similar to that of SUDV-infected humans and monkeys. We believe this model will significantly accelerate the development of life-saving measures against SUDV infections.

  • Baculovirus Inhibitor-of-Apoptosis Op-IAP3 Blocks Apoptosis by Interaction with and Stabilization of a Host Insect Cellular IAP [PublishAheadOfPrint]

  • Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda. Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its anti-apoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and auto-ubiquitination when bound to its targeted cellular IAP.

    IMPORTANCE The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, their anti-apoptotic mechanism in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, auto-ubiquitinating host IAP in such a way that cellular IAP levels and anti-apoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is the likely result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential has been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host-death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

  • Targeting swine SLA-I molecules for proteasomal degradation by the nsp1{alpha} replicase protein of the Chinese highly pathogenic PRRSV strain JXwn06 [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is a critical pathogen of swine, and the infections by this virus often result in delayed, low level induction of CTL responses in pigs. Here, we report that the Chinese highly pathogenic PRRSV possessed the ability to down-regulate swine leukocyte antigen class I (SLA-I) molecules on the cell surface of porcine alveolar macrophages and target them for degradation in a manner that was dependent on the ubiquitin-proteasome system. Moreover, we found that the nsp1aalpha; replicase protein contributed to this property of PRRSV. Further mutagenesis analyses revealed that this function of nsp1aalpha; required the intact molecule, including the zinc finger domain, but not the cysteine protease activity. More importantly, we found nsp1aalpha; was able to interact with both chains of SLA-I, a requirement that is commonly needed for many viral proteins to target their cellular substrates for proteasomal degradation. Together, our findings provide critical insights into the mechanisms of how PRRSV might evade cellular immunity, and also add a new role for nsp1aalpha; in PRRSV infection.

    IMPORTANCE PRRSV infections often result in delayed, low level induction of CTL responses in pigs. Deregulation of this immunity is thought to prevent the virus from clearance in an efficient and timely manner, contributing to persistent infections in swineherds. Our studies in this report provide critical insight into the mechanism of how PRRSV might evade CTL responses. In addition, our findings add a new role to nsp1aalpha;, a critical viral factor involved in antagonizing host innate immunity.

  • Identification of A Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus [PublishAheadOfPrint]

  • Over the past two decades, several novel influenza viral proteins have been identified that modulate viral infections in vitro and/or in vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. Here, we used RT-PCR targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, termed PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513nndash;1894 of the PB2 mRNA was deleted. PB2-S1 was detected in virus-infected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity depending on its PB1 binding capability. The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among human pre-2009 H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed similar growth kinetics in MDCK cells and virulence in mice to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation.

    IMPORTANCE Transcriptome analysis of cells infected with influenza A virus has improved our understanding of the host response to viral infection because such analysis yields considerable information about both in vitro and in vivo viral infection. However, little attention has been paid to transcriptomes derived from the viral genome. Here, we focused on the splicing of mRNA expressed from the PB2 segment and identified a spliced viral mRNA encoding a novel viral protein. This result suggests that other as-yet unidentified viral proteins encoded in spliced mRNAs could be expressed in virus-infected cells. A viral transcriptome, including a viral spliceosome, should be evaluated to gain new insights into influenza virus infection.

  • Gamma-delta T cells play a protective role in chikungunya virus-induced disease [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. As CHIKV is initially introduced into the skin where T cells are prevalent, we evaluated their response to CHIKV infection. CHIKV infection led to a significant increase in T cells in the infected foot and draining lymph node associated with the production of pro-inflammatory cytokines and chemokines in C57BL/6J mice. T cell-/- mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling compared to wild-type mice as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, T cell-/- mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint of versus wild-type mice which was independent of differences in CHIKV replication. These results suggest that T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage.

    IMPORTANCE: Recent epidemics including the 2004-2007 outbreak and the spread of CHIKV to naïve populations in the Caribbean, Central and South America with resultant cases imported into the U.S highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identify T cells as being important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics.

  • IFN-kappa inhibits HPV31 transcription by inducing Sp100 proteins [PublishAheadOfPrint]

  • High risk human papillomaviruses (hr-HPV) establish persistent infections in keratinocytes which can lead to cancer of the ano-genital tract. Interferons (IFNs) are a family of secreted cytokines that induce IFN stimulated genes (ISGs) of which many display antiviral activities. Transcriptome studies have indicated that established hr-HPV-positive cell lines display a reduced expression of ISGs which correlates with decreased levels of IFN-kappa, a type I IFN constitutively expressed in keratinocytes. Prior studies have also suggested that IFN-beta has anti-hr-HPV activity but the underlying mechanisms are not well understood. The downregulation of IFN-kappa by hr-HPV raises the possibility that IFN-kappa has anti-HPV activity. Using doxycycline-inducible IFN-kappa expression in CIN612-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrate that IFN-kappa inhibits the growth of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited 4-6h after IFN-kappa expression. This was also observed with recombinant IFN-beta suggesting a common mechanism of IFNs. RNA-seq analysis identified 1367 IFN-kappa regulated genes of which 221 were modulated ggt;2-fold. The majority of those (71%) matched known ISGs confirming that IFN-kappa acts as a bona fide type I IFN in hr-HPV-positive keratinocytes. RNAi and co-transfection experiments indicate that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-kappa. Consistent with published data, that Sp100 acts as a restriction factor for HPV18 infection, our results suggest that hr-HPV target IFN-kappa to prevent Sp100 expression and identify Sp100 as an ISG with anti-HPV activity.

    IMPORTANCE High risk HPV can establish persistent infections which may progress to ano-genital cancers. Hr-HPV interfere with the expression of interferon (IFN)-stimulated genes (ISG) which is due to reduced levels of IFN-kappa, an IFN that is constitutively expressed in human keratinocytes. This study reveals that IFN-kappa inhibits rapidly HPV transcription and that this is due to the induction of Sp100 proteins. Thus, Sp100 represents an ISG for hr-HPV.

  • Cytomegalovirus-specific CD4 T cells are cytolytic and mediate vaccine protection [PublishAheadOfPrint]

  • CD4 T cells provide protection against cytomegalovirus and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here we report the first two I-Ad restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes, and use a MHC-II tetramer to characterize their phenotype and function. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope- and organ-specific manner. In addition, CD4 T cell epitope vaccination of immune competent mice reduced MCMV replication in the same organs where CD4 CTL activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo.

    IMPORTANCE CD4 T cells mediate immune protection by using their T cell receptor to recognize specific portions of viral proteins, called epitopes, that are presented by major histocompatibility complex class-II (MHC-II) molecules on the surface of professional antigen presenting cells (APC). In this study we have discovered the first two epitopes derived from mouse cytomegalovirus (MCMV) that are recognized by CD4 T cells in BALB/c mice, a mouse strain commonly used to study the pathogenesis of this virus infection. Here we report the sequence of these epitopes, characterize the CD4 T cells that recognize them to fight off MCMV infection and show we can use these epitopes to vaccinate mice and protect against MCMV.

  • A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens [PublishAheadOfPrint]

  • In 1999, after circulation for a few months in poultry in Italy low pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS) PEIPKGSRVRR*GLF in the hemagglutinin (HA) and additional, alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 (Lp) and HPAI A/chicken/Italy/445/1999 (Hp) were generated. Hp with monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was substituted by that of Hp. In HA2, three amino acids differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental for replication in cell culture probably due to low pH-activation of HA. A436T and/or K536R restored viral replication in-vitro and in-vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but later superseded by viruses carrying all three mutations. Together, beside the mCS stepwise mutations in HA2 increased fitness of the Italian H7N1 virus in-vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization.

    IMPORTANCE In 1999, after 9 months of circulation of low pathogenic (LP) avian influenza virus a devastating highly pathogenic (HP) AIV H7N1 in poultry emerged marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main findings in this report are the identification of three mutations in the HA2 domain which are required for replication, stability as well as for virulence, transmission and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental for virus replication and required A436T and/or K536R to restore replication, systemic spread and stability. These results are important for better understanding the evolution of highly pathogenic avian influenza viruses from low pathogenic precursors.

  • Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and related viruses [PublishAheadOfPrint]

  • Viruses of the family Flaviviridae are important pathogens of humans and other animals, and currently classified into four genera. To better understand their diversity, evolutionary history and genomic flexibility, we used RNA-seq to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of 5 segmented Jingmenviruses and 12 distant relatives of the known Flaviviridae (llsquo;flavi-like' viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, while these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated.

    IMPORTANCE The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harbouring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses.

  • A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly [PublishAheadOfPrint]

  • Proteolytic maturation drives the conversion of stable, immature virus particles to a mature, metastable state primed for cell infection. In the case of human adenovirus, this proteolytic cleavage is mediated by the virally-encoded protease, AVP. Protein VI, an internal capsid cement protein and substrate for AVP, is cleaved at two sites, one of which is near the N-terminus of the protein. In mature capsids, the 33-residues at the N-terminus of protein VI (pVIn) are sequestered inside the cavity formed by peripentonal hexon trimers at the five-fold vertex. Here, we describe a glycine to alanine substitution in the N-terminal cleavage site of protein VI that profoundly impacts proteolytic processing, the generation of infectious particles, and cell entry. The phenotypic effects associated with this mutant provide a mechanistic framework for understanding the multifunctional nature of protein VI. Based on our findings, we propose that the primary function of the pVIn peptide is to mediate interactions between protein VI and hexon during virus replication, driving hexon nuclear accumulation and particle assembly. Once particles are assembled, AVP-mediated cleavage facilitates the release of the membrane lytic region at the amino terminus of mature VI, allowing it to lyse the endosome during cell infection. These findings highlight the importance of a single maturation cleavage site for both infectious particle production and cell entry, and emphasize the exquisite spatiotemporal regulation governing adenovirus assembly and disassembly.

    IMPORTANCE Post-assembly virus maturation is a cornerstone principle in virology. However, a mechanistic understanding of how icosahedral viruses utilize this process to transform immature capsids into infection-competent particles is largely lacking. Adenovirus maturation involves proteolytic processing of seven precursor proteins. There is currently no information for the role of each independent cleavage event in the generation of infectious virions. To address this, we investigated the proteolytic maturation of one adenovirus precursor molecule, protein VI. Structurally, protein VI cements the outer capsid shell and links it to the viral core. Functionally, protein VI is involved in endosome disruption, subcellular trafficking, transcription activation, and virus assembly. Our studies demonstrate that the multifunctional nature of protein VI is largely linked to its maturation. Through mutational analysis, we show that disrupting the N-terminal cleavage of pre-protein VI has major deleterious effects on the assembly of infectious virions and their subsequent ability to infect host cells.

  • Functional characterization of glycoprotein H chimeras composed of conserved domains of the pseudorabies virus and herpes simplex virus type 1 homologs [PublishAheadOfPrint]

  • Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein gB and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus type 2, Epstein-Barr-Virus, and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus type 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD and gL of PrV or HSV-1 were analysed. Chimeric gH containing domain I of HSV-1 and domains II-IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I-III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD and gL. Substitution of PrV gB by the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions.

    IMPORTANCE Envelope glycoprotein H (gH) is essential for herpesvirus induced membrane fusion, which is required for host cell entry and viral spread. Although gH is structurally conserved within the Herpesviridae, its precise role and its interactions with other components of the viral fusion machinery are not fully understood. Chimeric proteins containing domains of gH proteins from different herpesviruses can serve as tools to elucidate the molecular basis of gH function. The present study shows that the C-terminal part of Human herpesvirus 1 (herpes simplex virus type 1) gH can functionally substitute for the corresponding part of Suid herpesvirus 1 (pseudorabies virus) gH, whereas other tested combinations proved to be non-functional. Interestingly, the exchangeable fragment included the membrane-proximal end of the gH ectodomain (domain IV), which is most conserved in sequence and structure, and might be capable of transient membrane interaction during fusion.

  • Hepatocyte Heparan Sulfate is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo [PublishAheadOfPrint]

  • Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1HEP mutant mice exhibit an eight-fold reduction of heparan sulfate in primary hepatocytes and a five-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1HEP mice. However, in contrast to AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1HEP mice. We conclude that, while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study re-opens the question of how adenovirus enters cells in vivo.

    IMPORTANCE Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell-surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis is genetically abolished, demonstrate that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo, and for optimization of Ad5 vectors as therapeutic agents.

  • KSHV vIRF4 perturbs the G1-S cell cycle progression via deregulation of cyclin D1 [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the bbeta;-catenin/CBP cofactor and blocks its occupancy on cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.

  • Low-level expression of E1B 20K by Adenovirus type 14 p1 enhances viral immunopathogenesis [PublishAheadOfPrint]

  • Adenovirus (Ad) 14p1 is an emergent variant of Ad serotype14 (Ad14) that has caused an increased severity of respiratory illness during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased E1B 20K expression, when compared with infection with wild type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-B dependent, pro-inflammatory cytokine responses of responder human alveolar macrophages. A small animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.

    IMPORTANCE We reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-B-dependent macrophage cytokine responses (1). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1 infected human cells and that this causes an increased pro-inflammatory cytokine response of human alveolar macrophages and more severe inflammatory lung disease in infected hamsters. This is the first evidence of a clinical relevance of differential expression of the small Ad E1B gene product. The results suggest that there is a low, critical threshold of E1B 19/20K expression that is needed for viral replication and infection transmission but a higher level of E1B 19/20K expression required for the usual repression and control of the Ad-triggered host innate immune response.

  • Structure/Function Studies Involving the V3 Region of the HIV-1 Envelope Delineate Multiple Factors that Affect Neutralization Sensitivity [PublishAheadOfPrint]

  • Antibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most Tier 1 and many Tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans, and affected the nature of transmitted viruses in infected vaccinees despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is meta-stable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The "spring loaded" V3 in the envelope trimer is easily "released" by disruption of the stability of the "V3 pocket" in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is in turn dependent on the conformation of the bridging sheet, and on the stability of the V1V2 B-C strand-connecting loop.

    IMPORTANCE Antibodies levels to the third variable regions (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded as it sits in a pocket of the envelope trimer on the surface of virions, however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120: (a) render viruses sensitive to neutralization by V3 antibodies, (b) result in altered packing of the V3 loop, and (c) activate an "open" conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection, and why they should be one of the targets of vaccine-induced antibodies.

  • Growth and pathogenic potential of naturally selected reassortants after co-infection with pandemic H1N1 and HPAI H5N1 viruses [PublishAheadOfPrint]

  • Co-infected of ferrets with H5N1 and pH1N1 viruses resulted in two predominate-genotypes in the lungs containing surface gene/s of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice, but failed to transmit indirectly to naïve contact ferrets. Thus, this demonstrates a possible natural reassortment following co-infection as well as the pathogenicity of the potential reassortants.

  • Viral DNA-dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response, which, in turn, modulates viral gene expression and replication.

    IMPORTANCE Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can under selected conditions be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence.

  • Induction of Kaposi Sarcoma Herpesvirus-encoded Viral Interleukin-6 by X-Box Binding Protein-1 [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman disease (MCD). The KSHV life cycle has two principal gene repertoires, latent and lytic. KSHV viral interleukin (vIL-6), an analog of human IL-6, is usually lytic; production of vIL-6 by involved plasmablasts is a central feature of KSHV-MCD. vIL-6 also plays a role in PEL and KS. We show that a number of plasmablasts from lymph nodes of patients with KSHV-MCD express vIL-6 but not ORF45, a KSHV lytic gene. We further show that vIL-6 is directly induced by the spliced (active) X-box binding protein-1 (XBP-1s), a transcription factor activated by endoplasmic reticulum (ER) stress and differentiation of B cells in lymph nodes. The promoter region of vIL-6 contains several potential XBP-response elements (XRE), and two of these elements in particular mediate the effect of XBP-1s. Mutation of these elements abrogates the response to XBP-1s, but not to KSHV replication and transcription activator (RTA). Also, XBP-1s binds to the vIL-6 promoter in the region of these XRE. Exposure of PEL cells to a chemical inducer of XBP-1s can induce vIL-6 at low concentrations. Patient-derived PEL tumor cells that produce vIL-6 frequently co-express XBP-1, and immunofluorescence staining of involved KSHV-MCD lymph nodes reveals that most plasmablasts expressing vIL-6 also co-express XBP-1. These results provide evidence that XBP-1s is a direct activator of KSHV vIL-6 and that this is an important step in the pathogenesis of KSHV-MCD and PEL.

    IMPORTANCE Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD) is characterized by severe inflammatory symptoms caused by an excess of cytokines, particularly KSHV-encoded viral interleukin-6 (vIL-6) produced by lymph node plasmablasts. vIL-6 is usually a lytic gene. We show that a number of KSHV-MCD lymph node plasmablasts express vIL-6 but do not have full lytic KSHV replication. Differentiating lymph node B cells express spliced (active) X-box binding protein-1 (XBP-1s). We show that XBP-1s binds to the promoter of vIL-6 and can directly induces production of vIL-6 through X-box protein response elements on the vIL-6 promoter region. We further show that chemical inducers of XBP-1s can upregulate production of vIL-6. Finally, we show that most vIL-6-producing plasmablasts from lymph nodes of KSHV-MCD patients co-express XBP-1s. These results demonstrate that XBP-1s can directly induce vIL-6 and provide evidence that this is a key step in the pathogenesis of KSHV-MCD and other KSHV-induced diseases.

  • TRIM5 retroviral restriction activity correlates with the ability to induce innate immune signaling [PublishAheadOfPrint]

  • Host restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. But the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, b, and c, and Trim30a, b, c, and d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity each was fused to the HIV-1 CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions was inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling.

    IMPORTANCE The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. 8% of the human genome is retrovirus sequence, fixed in the germline during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to biology. TRIM5 is a cellular protein that protects host genome integrity by disrupting the retroviral capsid as it transports viral nucleic acid to the host cell nucleus. Previous data suggest that innate immune signaling contributes to TRIM5-mediated restriction. Here we show that activation of innate immune signaling is conserved among primate and carnivore TRIM5 orthologues, and among 3 of the 7 mouse Trim5 homologues, and that such activity is required for TRIM5-mediated restriction activity.

  • Comparative in vitro and in vivo studies of porcine rotavirus G9P[13] and human rotavirus Wa G1P[8] [PublishAheadOfPrint]

  • The changing epidemiology of group A rotavirus (RV) strains in humans and swine, including emerging G9 strains, poses new challenges to current vaccines. In this study, we comparatively assessed the pathogenesis of porcine RV (PRV) G9P[13] and evaluated the short-term cross-protection between this strain and human RV (HRV) Wa G1P[8] in gnotobiotic pigs. Complete genome sequencing demonstrated that PRV G9P[13] possessed a human-like G9 VP7 genotype but shared higher overall nucleotide identity with historic PRV strains. PRV G9P[13] induced longer rectal virus shedding and RV RNAemia in pigs than HRV Wa G1P[8] and generated complete short-term cross-protection in pigs challenged with HRV or PRV, whereas HRV Wa G1P[8] induced only partial protection against PRV challenge. Moreover, PRV G9P[13] replicated more extensively in porcine monocyte-derived dendritic cells (MoDCs) than HRV Wa G1P[8]. The cross-protection was likely not dependent on serum virus neutralizing (VN) antibodies as the heterologous VN antibody titers in the G9P[13]-inoculated pig sera were low. Thus, our results suggest that heterologous protection by the current monovalent G1P[8] HRV vaccine against the emerging G9 strains should be evaluated in clinical and experimental studies to prevent further dissemination of the G9 strains. Differences in the pathogenesis of these two strains may be partially attributable to their variable abilities to replicate and persist in porcine immune cells, including DCs. Additional studies are needed to evaluate the emerging G9 strains as potential vaccine candidates and to test the susceptibility of various immune cells to infection by G9 and other common HRV/PRV genotypes.

    IMPORTANCE The changing epidemiology of porcine and human group A rotaviruses (RV), including emerging G9 strains, may compromise the efficacy of current vaccines. Understanding the pathogenesis, genetic, immunological and biological features of the new emerging RV strains will contribute to development of new surveillance and prevention tools. Additionally, cross-protection studies within a susceptible host (swine) between the newly identified emerging G9 porcine RV strains and a human G1 RV vaccine strain will allow evaluation of the G9 strains as potential novel vaccine candidates to be included in porcine or human vaccines.

  • Kaposi's sarcoma-associated herpesvirus reduces cellular myeloid differentiation primary response gene-88 (MyD88) expression via modulation of its RNA [PublishAheadOfPrint]

  • Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human gamma herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Interleukin 1(IL1) is a major mediator for inflammation and plays an important role in both innate and adaptive immunity. Myeloid differentiation primary response gene 88 (MyD88) is an essential adaptor molecule for IL1 as well as most Toll-like receptors signaling. In this report, we identified a novel mechanism by which KSHV interferes with host inflammation and immunity. KSHV RTA specifically reduces the steady state protein levels of MyD88, and physiological levels of MyD88 are down regulated during KSHV lytic replication when RTA is expressed. The N-terminal region of RTA is required for the reduction of MyD88. Additional studies demonstrated that RTA targets MyD88 expression at the RNA level, inhibits RNA synthesis of MyD88, and may bind MyD88 RNA. Finally, RTA inhibits interleukin 1(IL1)-mediated activation of NF-kappaB (NF-B). Because IL1 is abundant in KS microenvironment and inhibits KSHV replication, this work may expand our understanding on how KSHV evades host inflammation and immunity for its survival in vivo.

    IMPORTANCE MyD88 is an important molecules for IL1 mediated inflammation and TLR signaling. This work shows that KSHV inhibits MyD88 expression through a novel mechanism. KSHV RTA may bind to MyD88 RNA, suppresses RNA synthesis of MyD88, and inhibits IL1-mediated signaling. This work may expand our understanding on how KSHV evades host inflammation and immunity.

  • Identification of MEF2B, EBF1, and IL6R as direct gene targets of EBNA1 critical for EBV-infected B-lymphocyte survival [PublishAheadOfPrint]

  • EBNA1 is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin-binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome, and can provide a host cell survival function, but it is not yet know how EBNA1-sequence specific binding is responsible for host cell survival. Here, we integrate EBNA1 ChIP-Seq with RNA-Seq after EBNA1-depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV positive cells, including Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and lymphoblastoid cell lines (LCL). EBNA1 binds to ~1000 sites that are mostly invariant among cell types and share a common consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation, and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1-depletion by decreasing cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability, and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells.

    Significance Statement

    Epstein-Barr Virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is consistently expressed in all EBV-associated cancers. EBNA1 is known to provide a host-cell survival function, but the mechanism is not known. EBNA1 is a sequence specific binding protein important for viral genome maintenance during latency. Here, we demonstrate by integrating ChIP-Seq and RNA-Seq that EBNA1 binds directly to the promoter regulatory regions and up-regulates transcription of host genes that are important for the survival of EBV-infected cells. Identification of EBNA1 target genes provides potential new targets for therapeutic intervention in EBV associated disease.

  • M2 macrophages play critical roles in progression of inflammatory liver disease in hepatitis C virus transgenic mice [PublishAheadOfPrint]

  • Macrophages in liver tissue are widely defined as important inflammatory cells in chronic viral hepatitis due to their pro-inflammatory activity. We reported previously that IL-6 and TNF-aalpha; play significant roles in causing chronic hepatitis in hepatitis C virus (HCV) transgenic mice. In addition, we showed that recombinant vaccinia viruses expressing a HCV nonstructural protein (rVV-N25) could protect against the progression of chronic hepatitis by suppression of macrophage activation. Herein we focus on the role of macrophages in liver disease progression in HCV transgenic mice and examine characteristic features of macrophages following rVV-N25 treatment. The number of CD11b+F4/80+CD11c-CD206+ (M2) macrophages in the liver of HCV transgenic mice was notably increased compared to that of age-matched control mice. These M2 macrophages in the liver produced elevated levels of IL-6 and TNF-aalpha;. rVV-N25 infection suppressed the number and activation of M2 macrophages in liver tissue. These results suggested that inflammatory cytokines produced by M2-like macrophages contribute to the induction of chronic liver inflammation in HCV transgenic mice. Moreover, the therapeutic effect of rVV-N25 might be induced by suppression of the number and activation of hepatic macrophages.

    IMPORTANCE HCV causes persistent infections that can lead to chronic liver diseases, liver fibrosis, and hepatocellular carcinoma; the search for an HCV curative is the focus of ongoing research. Recently, effective anti-HCV drugs have been developed; however, vaccine development is still required for prevention and therapy of infection by this virus. We demonstrate here that M2 macrophages are important for the pathogenesis of HCV-caused liver diseases, and additionally show that M2 macrophages contribute to the therapeutic mechanism observed following rVV-N25 treatment.

  • Parameters of Mosquito-Enhanced West Nile Virus Infection [PublishAheadOfPrint]

  • The arthropod-borne West Nile virus (WNV) emerged in New York in 1999 and quickly spread throughout the United States. Transmission is maintained in an enzootic cycle in which infected mosquitoes transmit the virus to susceptible hosts during probing and feeding. Arthropod-derived components within the viral inoculum are increasingly acknowledged to play a role in infection of vertebrate hosts. Our laboratory previously showed that Culex tarsalis mosquito saliva and salivary gland extract (SGE) enhance in vivo replication of WNV. Here, we characterized the effective dose, timing, and proximity of saliva and SGE administration necessary for enhancement of WNV viremia, using a mouse model. Mosquito saliva and SGE enhanced viremia in a dose-dependent manner, and a single mosquito bite or as little as 0.01 mmu;g of SGE were effective, suggesting a potent active salivary factor. Viremia was enhanced when SGE was injected in the same location 24 hours before virus inoculation through 12 hours after virus inoculation. These results were confirmed with mosquito saliva deposited by uninfected mosquitoes. When salivary treatment and virus inoculation were spatially separated, viremia was not enhanced. In summary, the effects of mosquito saliva and SGE were potent, long-lasting, and localized, and these studies have implications for virus transmission in nature where vertebrate hosts are fed upon by both infected and uninfected mosquitoes over time. Furthermore, our model will provide a robust system to identify the salivary factor(s) responsible for enhancement of WNV replication.

    Importance Mosquito-borne viruses are a significant class of emerging infectious disease. WNV has caused over 18,000 cases of neuroinvasive disease in the United States since its emergence. Our laboratory has shown that Culex tarsalis mosquito saliva and SGE enhance replication of WNV. We now demonstrate that saliva and SGE have potent, long-lasting, and localized effects. Our model will provide a robust system to identify the salivary factor(s) and characterize the mechanism responsible for enhancement of WNV replication. These studies could lead to the identification of novel prophylactic or treatment options useful in limiting the spread and disease of WNV and other mosquito-borne viruses.

  • IRF-5-dependent signaling restricts Orthobunyavirus dissemination to the central nervous system [PublishAheadOfPrint]

  • Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3, Irf5, and Irf7 or Irf5 alone. Deletion of Irf3, Irf5, and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, one to two weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5-/- mice were associated with abundant viral antigen and TUNEL-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5-/- mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN compared to WT cells. This result was supported by data obtained in vitro, since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice.

    IMPORTANCE Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3-/- Irf7-/- (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar-/- mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV.

  • The dynamic response of IFI16 and PML Nuclear Body components to HSV-1 infection. [PublishAheadOfPrint]

  • Intrinsic immunity is an aspect of antiviral defence that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus the balance between cellular repressors and viral counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus type 1 (HSV-1) is conferred by components of PML Nuclear Bodies, which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins also respond to viral genomes in this manner, including IFI16 which has been implicating in sensing pathogen DNA and initiating signalling pathways that lead to an interferon response. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrate that this response is extremely rapid, occuring within the first hour after addition of the virus, and that hDaxx and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral genome induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0.

    IMPORTANCE Herpesviruses have intimate interactions with their hosts, with infection leading either to the productive lytic cycle or to a quiescent infection in which viral gene expression is suppressed while the viral genome is maintained in the host cell nucleus. Whether a cell becomes lytically or quiescently infected can be determined through the competing activities of cellular repressors and viral activators, some of which counteract cell mediated repression. Therefore the events that occur within the earliest stages of infection can be of crucial importance. Using live cell microscopy, this paper describes the extremely rapid response to herpes simplex virus type 1 infection of the cellular protein IFI16, a sensor of pathogen DNA, and also the PML Nuclear Body proteins PML and hDaxx. The data imply that these proteins can accumulate on or close to the viral genomes in a sequential manner which may lead to their sequenstration and repression.

  • The missing link in EBV immune evasion: the BDLF3 gene induces ubiquitination and downregulation of MHC class I and MHC class II [PublishAheadOfPrint]

  • The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune-evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is in part due to the early EBV protein, BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune-evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic gene, BDLF3, as the missing link accounting for the efficient evasion during late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 down-regulates expression of surface MHC class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (Transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasomal dependent manner.

    IMPORTANCE Immune-evasion is a necessary feature of viruses that establish life-long persistent infections in the face of strong immune-responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune-evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically-infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune-evasion protein, BDLF3 that is expressed late in lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasomal dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules, to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral gene that impairs both the class I and class II MHC antigen presenting pathways.

  • Macaque monoclonal antibodies targeting novel conserved epitopes within filovirus glycoprotein [PublishAheadOfPrint]

  • Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan (SUDV), Bundibugyo (BDBV), and Marburg (MARV) viruses have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GP) and virus-like particles (VLPs) for three divergent filovirus species. The antibodies recognize novel neutralizing and non-neutralizing epitopes on the filovirus glycoprotein including conserved conformational epitopes within the core regions of GP1 subunit as well a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP, cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered at two doses and partial, but significant, protection when given once at the peak of viremia 3 days post infection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual mAbs. In summary, the present work describes multiple novel, cross reactive filovirus epitopes as well as innovative combination concepts that challenge the current therapeutic models.

    IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to over 27,000 cases and 11,000 fatalities. While there are five species of ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus (EBOV). Since the nature of future outbreaks cannot be predicted there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

  • Pan-ebolavirus and pan-filovirus mouse monoclonal antibodies: protection against Ebola and Sudan viruses [PublishAheadOfPrint]

  • The unprecedented 2014-15 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (mAb) cocktails have shown great potential as EVD therapeutics, however, the existing protective mAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses towards conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses as well as a pan-filovirus antibody reactive to the receptor binding region of all filovirus glycoproteins. Significant post-exposure efficacy of several mAbs including a novel antibody cocktail was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species; i.e. Ebola and Sudan viruses, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species.

    IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the US biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV; formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola and Sudan viruses in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

  • Interaction Between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex is Essential For Viral Late Gene Expression [PublishAheadOfPrint]

  • Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in -herpesviruses (-HV) requires coordination between six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus (KSHV). Disruption of this complex via point mutation of the interaction interface between the ORF24 and ORF34 vTFs ablated both late gene expression and viral replication.

  • Single Genome Sequencing of Hepatitis C Virus in Donor-Recipient Pairs Distinguishes Modes and Models of Virus Transmission and Early Diversification [PublishAheadOfPrint]

  • Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single genome sequencing of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pre-transmission bottleneck due to treatment, immune selection or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences.

    Importance Despite the recent development of highly effective, interferon-sparing anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and the development of an effective vaccine, which could be facilitated by a precise molecular identification of transmitted/founder (T/F) viral genomes and their progeny. We used single genome sequencing to show that inferred HCV T/F sequences in recipients were identical to viral sequences in their respective donors and that viral genomes generally evolved early in infection according to a simple model of random sequence evolution. Altogether, the findings validate T/F genome inferences and illustrate how T/F sequence identification can illuminate studies of HCV transmission, immunopathogenesis, drug resistance development and vaccine protection, including sieving effects on breakthrough virus strains.

  • The Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (kRSV-A2GA2F and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection.

    IMPORTANCE RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. Like other paramyxoviruses, two major RSV surface viral glycoproteins, the attachment protein G and the fusion protein F, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We re-evaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

  • Crystal structure of the conserved amino-terminus of the extracellular domain of matrix protein 2 of influenza A virus gripped by an antibody [PublishAheadOfPrint]

  • We report the crystal structure of the M2 ectodomain (M2e) in complex with a monoclonal antibody that binds the amino-terminus of M2. M2e stretches out into the antibody binding site to form an N-terminal bbeta;-turn near the bottom of the paratope. This M2e folding differs significantly from M2e in complex with an antibody that binds another part of M2e. This suggests that M2e can adopt at least two conformations that can elicit protective antibodies.

  • The critical role of K1685 and K1829 in the large protein of rabies virus on viral pathogenicity and immune evasion [PublishAheadOfPrint]

  • Rabies, one of the oldest infectious diseases, still presents a public health threat today in most parts of the world. Its pathogen, rabies virus (RABV), could utilize its viral proteins, such as nucleoprotein and phosphorylation protein to subvert the host innate immune system. For a long time, the large (L) protein was believed to be essential for RABV transcription and replication, but its role on viral pathogenicity and immune evasion was not known. According to recent studies, the conserved K-D-K-E tetrad motif in the L protein was found to be related to the methyltransferase (MTase) activity in the viral mRNA process. In this present study, a series of RABV mutations in this motif was constructed with the recombinant CVS-B2c (rB2c) virus. Two of these mutants, rB2c-K1685A and rB2c-K1829A, were found to be stable and displayed an attenuated phenotype in both in vitro growth and in vivo pathogenicity in adult and suckling mice. Further studies demonstrated that these two mutants were more sensitive to the expression of the interferon stimulated gene IFIT2. Taken together, our results suggest that K1685 and K1829 in the L protein play an important role in the pathogenicity and immune evasion during RABV infection.

    IMPORTANCE Rabies continues to present a public health threat in most areas of the world, especially in the developing countries of Asia and Africa. The pathogenic mechanisms for rabies are not well understood. In the present study, it was found that the recombinant rabies virus rB2c-K1685A and rB2c-K1829A, carrying a mutation at the predicted MTase catalytic sites in the L protein, were highly attenuated both in vitro and in vivo. Further studies showed that these mutants were more sensitive to the expression of the interferon stimulated gene IFIT2 than the parent virus. These findings provide a better understanding of rabies pathogenesis, which may help in developing potential therapeutics and avirulent rabies vaccine.

  • Natural Killer Cells and Innate IFN-{gamma} Participate in Host Defense against Respiratory Vaccinia Virus Infection [PublishAheadOfPrint]

  • In establishing a respiratory infection, vaccinia virus (VACV) initially replicates in airway epithelial cells before spreading to secondary sites of infection, mainly the draining lymph nodes, spleen, gastrointestinal tract, and reproductive organs. We recently reported that IFN- produced by CD8 T cells ultimately controls this disseminated infection, but the relative contribution of IFN- early in infection is unknown. Investigating the role of innate cells, we found that the frequency of natural killer (NK) cells in the lung increased dramatically between days 1 and 4 post-infection with VACV. Lung NK cells displayed an activated cell surface phenotype, and were the primary source of IFN- prior to arrival of CD8 T cells. In the presence of an intact CD8 T cell compartment, depletion of NK cells resulted in increased lung viral load at the time of peak disease severity, but had no effect on eventual viral clearance, disease symptoms, or survival. In sharp contrast, RAG-/- mice devoid of T cells failed to control VACV and succumbed to infection despite a marked increase in NK cells in the lung. Supporting an innate role for NK-derived IFN-, we found that NK-depleted or IFN--depleted RAG-/- mice displayed increased lung VACV titers and dissemination to ovaries and a significantly shorter mean time to death compared to untreated NK-competent RAG-/- controls. Together, these findings demonstrate a role for IFN- in aspects of both the innate and adaptive immune response to VACV, and highlight the importance of NK cells in T-independent control of VACV in the respiratory tract.

    IMPORTANCE Herein, we provide the first systematic evaluation of natural killer (NK) cell function in the lung after infection with vaccinia virus, a member of the Poxviridae family. The respiratory tract is an important mucosal site for entry of many human pathogens including poxviruses, but precisely how our immune system defends the lung against these invaders remains unclear. Natural killer cells are a type of cytotoxic lymphocyte and part of our innate immune system. In recent years, NK cells have received increasing levels of attention following the discovery that different tissues contain specific subsets of NK cells with distinctive phenotypes and function. They are abundant in the lung but their role in defense against respiratory viruses is poorly understood. What this study demonstrates is that NK cells are recruited, activated and contribute to protection of the lung during a severe respiratory infection with vaccinia virus.

  • The phosphatidylserine and phosphatidylethanolamine receptor CD300a binds dengue virus and enhances infection [PublishAheadOfPrint]

  • Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PdtSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PdtSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PdtEth and PdtSer present on virions and enhance infection.

    IMPORTANCE Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300anndash;dependent DENV infection relies on the direct recognition of PtdEth and to a lesser extent PtdSer associated with viral particles. This study provides novel insights into the mechanisms that mediate DENV entry and reinforce the concept that DENV uses an apoptotic mimicry strategy for viral entry.


  • Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two pore Ca2+ channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging we provide evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.

  • A new member of the V1V2-directed CAP256-VRC26 lineage that shows increased breadth and exceptional potency [PublishAheadOfPrint]

  • The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of twelve V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al, Nature 50:55-62, 2014 doi:10.1038/nature13036). Here, we report the isolation and characterization of new members of the family, mostly from time points of peak serum breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed 10-fold greater neutralization potency than previous published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among viruses neutralized, the median IC50 was 0.001 micrograms per ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage, and describe more potent and broadly neutralizing members with potential clinical utility, particularly in clade C-prevalent areas.

    IMPORTANCE Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C infected donor. Here, we report the isolation and characterization of new members of the family, mostly from time points of peak serum breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed 10-fold greater neutralization potency than previous described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: a median IC50 of 0.001 micrograms per ml. Our results highlight the ongoing evolution within a single antibody lineage, and describe more potent and broadly neutralizing members with potential clinical utility, particularly in clade C-prevalent areas.

  • Induction of broad-based immunity and protective efficacy by self-amplifying mRNA vaccines encoding influenza virus hemagglutinin [PublishAheadOfPrint]

  • Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immune-compromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza strains combine with seasonal strains. In this study, we used the SAMrreg; technology and characterized the immunogenicity and efficacy of a self-amplifying RNA expressing influenza hemagglutinin (HA) antigen, (SAM(HA)) formulated with a novel oil-in-water cationic nanoemulsion (CNE). We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HAnndash;specific CD4 T helper (Th) 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza.

    IMPORTANCE In this paper, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza. This novel type of vaccine elicits both humoral and cellular immune responses. While vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make this platform particularly exploitable in case of influenza pandemic.

  • Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase [PublishAheadOfPrint]

  • Antibodies against the neuraminidase (NA) of influenza virus correlate with resistance against disease, but the effectiveness of antibodies against different NA epitopes has not been compared. In the present study, we evaluated the in vitro and in vivo efficacy of four monoclonal antibodies (MAbs): HF5 and CD6 that are specific to two different epitopes in the NA of 2009 pandemic H1N1 (pH1N1) virus, and 4E9 and 1H5 that are specific to a conserved epitope in the NA of both H1N1 and H5N1viruses. In the in vitro assays, HF5 and CD6 inhibited virus spread and growth more effectively than 4E9 and 1H5, with HF5 being the most effective inhibitor. When administered prophylactically at 5 mg/kg, HF5 and CD6 protected ~90-100% of DBA/2 mice against lethal wild-type pH1N1 virus challenge; however, at a lower dose (1 mg/kg), HF5 protected ~90% of mice, whereas CD6 only protected 25%. 4E9 and 1H5 were less effective than HF5 and CD6, as indicated by the partial protection achieved even at doses as high as 15 mg/kg. When administered therapeutically, HF5 protected a greater proportion of mice against lethal pH1N1 challenge than CD6. However, HF5 quickly selected pH1N1 virus escape mutants in both prophylactic and therapeutic treatments, while CD6 did not. Our findings confirm the important role of NA-specific antibodies in influenza immunity, and provide insight into the properties of NA antibodies that may serve as good candidates for therapeutics against influenza.

    IMPORTANCE Neuraminidase (NA) is one of the major surface proteins of influenza virus, serving as an important target for antivirals and therapeutic antibodies. The impact of NA-specific antibodies on NA activity and virus replication is likely to depend on where the antibody binds. Using in vitro assays and the mouse model, we compared the inhibitory/protective efficacy of four mouse monoclonal antibodies (MAbs) that bind to different sites within the 2009 pandemic H1N1 (pH1N1) virus NA. The ability of each MAb to protect mice against lethal pH1N1 infection corresponded to its ability to inhibit NA activity in vitro, however the MAb that was the most effective inhibitor of NA activity selected pH1N1 escape variants in vivo. One of the tested MAbs, which binds to a conserved region in the NA of pH1N1 virus, inhibits NA activity but did not result in escape variants, highlighting its suitability for therapeutic agent development.

  • The Hepatitis C Virus NS4B Can Suppress STING Accumulation to Evade Innate Immune Responses [PublishAheadOfPrint]

  • The cyclic dinucleotide 2rrsquo;, 3rrsquo; -cGAMP can bind the adaptor protein STING (Stimulator of interferon genes) to activate the production of type I interferons (IFNs) and proinflammatory cytokines. We found that cGAMP added to the culture media could suppress the replication of hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication while over-expression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP when compared to that of 1b/Con1 replicon. Using chimeric replicons, the 2a NS4B was identified to confer the resistance to cGAMP. Transient expression of the 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated IFN-bbeta; reporter activation when compared to that of the 1b NS4B. The 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of the 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response.

    Importance: The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon, but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in the 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N-terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that the 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation.

  • Duck IFITM3 mediates restriction of influenza viruses [PublishAheadOfPrint]

  • Interferon Inducible Transmembrane Proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV), and several other viruses that also enter host cells through the endocytic pathway. Here we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza. We identify and sequence duck IFITM1, IFITM2, IFITM3 and IFITM5. Using qPCR we demonstrate upregulation of these genes in lung tissue in response to highly pathogenic IAV infection 400-fold, 30-fold, 30-fold and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1 containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2 and H11N9 IAV strains, but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function, however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXX endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not the sole contributor to correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to host-viral co-evolution, duck IFITM3 is functional against IAV.

    IMPORTANCE Immune IFITM genes are poorly conserved across species suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether co-evolution between viruses and their natural host would result in evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses (IAV) and display little to no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus, and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza. Thus, despite long co-evolution of virus and host effectors in the natural host, influenza evasion of IFITM3 restriction in ducks is not apparent.

  • PB1-F2 Protein does not impact the Virulence of Triple Reassortant H3N2 Swine Influenza Virus in Pigs but Alters the Pathogenicity and Transmission in Turkeys [PublishAheadOfPrint]

  • PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and post-influenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host such as swine and avian hosts such as turkeys remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145) wild type (WT), PB1-F2 knock out mutant (SIV 1145-KO) and its N66S variant (SIV 1145-N66S). Ablation of PB1-F2 in SIV 1145 modulated early stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed higher histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. Higher histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO virus need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV.

    IMPORTANCE Novel triple reassortant H3N2 swine influenza virus (TR H3N2 SIV) emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed increased propensity to reassort generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts such as swine and turkeys and highlights the host-specific role of PB1-F2 protein.

  • pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold to Provide Tension for Membrane Fusion. [PublishAheadOfPrint]

  • Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At pH ~ 6, M1 interacts with the viral ribonuclear protein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to pH ~ 5 to clear a physical path for the viral genome to transit from the viral interior to cytoplasm. Here we investigate the physico-chemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The interaction energy of M1 molecules to each other in M1 dimers was about ten-fold weaker than M1 to lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in M1 charge, leading to alterations in their electrostatic interactions. Dropping pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1, due to increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of vesicle membrane, presumably due to the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release.

    IMPORTANCE Influenza remains a top killer of human beings throughout the world, in part due to rapid binding and uptake by cells into compartments hidden from the immune system. To attack influenza during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs.

  • Sabin vaccine reversion in the field: a comprehensive analysis of Sabin-like poliovirus isolates in Nigeria [PublishAheadOfPrint]

  • To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the wild-type rate due to increased non-synonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like evolution with the wild-type circulating VP1 molecular clock to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for type 1, 210 days for type 2, and 390 days for type 3. Phylogenetic relationships indicated transient local transmission of Sabin 3 and possibly Sabin 1 during periods of low wild polio incidence. Comparison of Sabin-like recombinants with known Nigerian VDPV recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin-Sabin and Sabin-non-Sabin enterovirus recombination after accounting for time from dose to detection. Our study provides a comprehensive picture of the evolutionary dynamics of oral polio vaccine in the field.

    IMPORTANCE The global polio eradication effort has completed its twenty-sixth year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed for vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived poliovirus (cVDPV) that have properties indistinguishable from wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in oral polio vaccine (OPV) in populations that have low immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about OPV evolution in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about polio intra-host dynamics to provide quantitative insight into polio vaccine evolution in the field.

  • The C-type lectin langerin functions as a receptor for attachment and infectious entry of influenza A virus [PublishAheadOfPrint]

  • It is well established that IAV attachment to and infection of epithelial cells is dependent on cell-surface sialic acid (SIA), although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA-deficient and resistant to IAV infection. Herein, we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral messenger (m)RNA and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently, but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH- and dynamin-dependent, occurred via clathrin- and caveolin-mediated endocytic pathways and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell-surface receptor.

    IMPORTANCE On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Herein, we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by a SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin.

  • Proton channel activity of influenza A virus matrix protein 2 contributes to autophagy arrest [PublishAheadOfPrint]

  • Influenza A virus infection can arrest autophagy as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosome with lysosome, which might be a key mechanism for arresting autophagy.

  • A structurally unresolved head segment of defined length favors proper measles virus hemagglutinin tetramerization and efficient membrane fusion triggering [PublishAheadOfPrint]

  • Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin, H) tetramer and a fusion protein (F) trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or co-crystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport, or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion-trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high molecular weight H-oligomers. Some oligomers had reduced fusion-trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H-tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers.

    IMPORTANCE Understanding the molecular mechanism of membrane fusion triggering may allow developing new antiviral strategies. The fusion apparatus of Paramyxoviruses consists of a receptor-binding tetramer and a fusion protein trimer. Structural analyses of the receptor-binding hemagglutinin-neuraminidase of certain Paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H-heads on a leash long enough to adopt a triggering-permissive conformation, but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a "leash", suggesting a conserved triggering mechanism.

  • Oligomerization requirements for MX2 mediated suppression of HIV-1 infection [PublishAheadOfPrint]

  • Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG), and was recently identified as a late post-entry suppressor of human immunodeficiency virus type-1 (HIV-1) infection, inhibiting nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2 mediated inhibition, the precise mechanism of anti-viral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related, and share a similar domain architecture and structure. However, MX2 possesses an extended N-terminus that is essential for anti-viral function and confers anti-HIV-1 activity on MX1 (MX1(NMX2)). Higher order oligomerization is required for the anti-viral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking, and co-immunoprecipitation. We show that, while monomeric MX2 and MX1(NMX2) mutants are not anti-viral, higher order oligomerization does not appear to be required for full anti-viral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for effective inhibition of HIV-1.

    IMPORTANCE Interferon (IFN) plays an important role in the control of virus replication during acute infection in vivo. Recently, cultured cell experiments identified human myxovirus resistance 2 (MX2/MXB) as a key effector in the interferon mediated post-entry block to human immunodeficiency virus type-1 (HIV-1) infection. MX2 is a member of a family of large dynamin-like GTPases that includes MX1/MXA, a closely related interferon inducible inhibitor of several viruses including influenza A virus (FLUAV), but not HIV-1. MX GTPases form higher-order oligomeric structures, and the oligomerization of MX1 is required for inhibitory activity against many of its viral targets. Through structure-function studies, we report that monomeric mutants of MX2 do not inhibit HIV-1. However, in contrast to MX1, oligomerization beyond dimer assembly does not seem to be required for the anti-viral activity of MX2 implying that fundamental differences exist between the anti-viral mechanisms employed by these closely related proteins.

  • Characterization and Demonstration of value of a Lethal Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease [PublishAheadOfPrint]

  • Characterized animal models are needed for studying pathogenesis of and evaluating medical countermeasures for the persisting Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) infection. Here, we further characterized a lethal transgenic mouse model of MERS-CoV infection and disease that globally expresses hCD26/DPP4. The 50% infectious dose (ID50) and lethal dose (LD50) of virus were estimated to be llt;1 and 10 TCID50 of MERS-CoV, respectively. Neutralizing antibody developed in surviving mice from the ID50/LD50 determinations and all were fully immune to challenge with 100 LD50 of MERS-CoV. The tissue distribution and histopathology in mice challenged with a potential working dose of 10 LD50 of MERS-CoV was subsequently evaluated. In contrast to the overwhelming infection in mice challenged with 105 LD50 of MERS-CoV, we were only able to infrequently recover infectious virus from these mice although qRTPCR tests indicated early and persistent lung infection and delayed occurrence of brain infection. Persistent inflammatory infiltrates were seen in the lungs and brain stems at day 2 and day 6 after infection, respectively. While focal infiltrates were also noted in the liver, definite pathology was not seen in other tissues. Finally, using a receptor binding domain protein vaccine and a MERS-CoV fusion inhibitor, we demonstrated the value of this model for evaluating vaccines and antivirals against MERS. As outcomes of MERS-CoV infection in patients differ greatly, ranging from asymptomatic to overwhelming disease and death, having available both an infection and a lethal model makes this transgenic mouse model relevant for advancing MERS research.

    IMPORTANCE Fully characterized animal models are essential for studying pathogenesis and for preclinical screening of vaccines and drugs against MERS-CoV infection and disease. When given a high-dose of MERS-CoV, our transgenic mice expressing hCD26/DPP4 viral receptor uniformly succumbed to death within 6 days, making it difficult to evaluate host responses to infection and disease. We further characterized this model by determining both the ID50 and LD50 doses of MERS-CoV in order to establish both an infection and a lethal model for MERS and followed this by investigating antibody responses and immunity of mice survived from MERS-CoV infection. Using the estimated LD50 and ID50 doses, we dissected the kinetics of viral tissue distribution and pathology in mice challenged with 10 LD50 of virus, and utilized the model for preclinical evaluation of a vaccine and drug for MERS-CoV infection. This further characterized transgenic mouse model will be useful for advancing MERS research.

  • Distinct escape pathway by HCV genotype 1a from a dominant CD8+ T cell response by selection of altered epitope processing [PublishAheadOfPrint]

  • Antiviral CD8+ T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by pre-existing viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8+ T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI1373-1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8+ T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, detection of strong CD8+ T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues up-stream of the epitope (S1368P; p=0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses.

    IMPORTANCE HCV is able to evolutionary adapt to CD8+ T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids upstream of the epitope underlines that efficient antigen presentation strongly depends on its larger sequence context and that blocking of the multi-step process of antigen processing by mutation is exploited also by HCV. The pathways to mutational escape of HCV are to some extent predictable but are distinct in different genotypes. Importantly, the selected escape pathway of HCV may have consequences for the destiny of antigen-specific CD8+ T cells.

  • Human Intestinal Enteroids: A New Model to Study Human Rotavirus Infection, Host Restriction and Pathophysiology [PublishAheadOfPrint]

  • Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes, goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these non-transformed, 3D HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs as they generally replicate poorly in transformed cell lines and host range restriction prevents their replication in many animal models whereas many animal RVs (ARV) exhibit a broader host range and replicate in mice. Using HRVs, including the RV1 Rotarixttrade; vaccine strain and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than ARV. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused a physiologic lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model, which will allow the study of HRV biology including host restriction, cell-type restriction, and viral-induced fluid secretion.

    IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology, pathophysiology and the epithelial response, including host restriction to gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children lle; age five. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, diverse cellular and physiologically relevant epithelial cultures, which recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions, host and cell-type restriction, viral-induced fluid secretion, cell-cell communication within the epithelium, and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures.

  • Genome-wide gene expression analysis identifies the proto-oncogene tyrosine-protein kinase Src as a crucial virulence determinant of the infectious laryngotracheitis virus in chicken cells [PublishAheadOfPrint]

  • Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) as an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo, but is not required for ILTV-induced cell death. Furthermore, our results identify a positive feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK, and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy.

    IMPORTANCE Despite the extensive administration of live attenuated vaccines starting from the mid-twentieth century, and the administration of recombinant vaccines in recent years, infectious laryngotracheitis (ILT) outbreaks due to avian ILT herpesvirus (ILTV) occur worldwide annually. Presently, there are no drugs or control strategies that effectively treat ILT. Targeting host-virus interactions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level, and provide experimental evidence that it is possible to control ILT via the manipulation of host-virus interactions.

  • Mismatches in Influenza A virus vRNA panhandle prevent RIG-I sensing by impairing RNA/RIG-I complex formation [PublishAheadOfPrint]

  • Influenza virus RNA promoter panhandle structures are believed to be sensed by RIG-I. The occurrence of mismatches in this dsRNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation, and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

  • Crawling with virus: translational insights of infant RSV pathogenesis from neonatal mice [PublishAheadOfPrint]

  • The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. Organized by cell type, we describe numerous age-specific responses observed in RSV-infected neonatal mice, and draw comparisons (when possible) to human infants.

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