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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]

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

  • Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection [Gems]

  • It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication.

  • Going Viral with Fluorescent Proteins [Gems]

  • Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  • Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities [Cellular Response to Infection]

  • Measles virus (MeV) causes several unique syndromes, including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase, which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions.

    IMPORTANCE Measles virus (MeV) is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by cellular protein phosphatase 5 (PP5) and DNA-dependent protein kinase. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV.

  • Circulative Nonpropagative Aphid Transmission of Nanoviruses: an Oversimplified View [Genome Replication and Regulation of Viral Gene Expression]

  • Plant virus species of the family Nanoviridae have segmented genomes with the highest known number of segments encapsidated individually. They thus likely represent the most extreme case of the so-called multipartite, or multicomponent, viruses. All species of the family are believed to be transmitted in a circulative nonpropagative manner by aphid vectors, meaning that the virus simply crosses cellular barriers within the aphid body, from the gut to the salivary glands, without replicating or even expressing any of its genes. However, this assumption is largely based on analogy with the transmission of other plant viruses, such as geminiviruses or luteoviruses, and the details of the molecular and cellular interactions between aphids and nanoviruses are poorly investigated. When comparing the relative frequencies of the eight genome segments in populations of the species Faba bean necrotic stunt virus (FBNSV) (genus Nanovirus) within host plants and within aphid vectors fed on these plants, we unexpectedly found evidence of reproducible changes in the frequencies of some specific segments. We further show that these changes occur within the gut during early stages of the virus cycle in the aphid and not later, when the virus is translocated into the salivary glands. This peculiar observation, which was similarly confirmed in three aphid vector species, Acyrthosiphon pisum, Aphis craccivora, and Myzus persicae, calls for revisiting of the mechanisms of nanovirus transmission. It reveals an unexpected intimate interaction that may not fit the canonical circulative nonpropagative transmission.

    IMPORTANCE A specific mode of interaction between viruses and arthropod vectors has been extensively described in plant viruses in the three families Luteoviridae, Geminiviridae, and Nanoviridae, but never in arboviruses of animals. This so-called circulative nonpropagative transmission contrasts with the classical biological transmission of animal arboviruses in that the corresponding viruses are thought to cross the vector cellular barriers, from the gut lumen to the hemolymph and to the salivary glands, without expressing any of their genes and without replicating. By monitoring the genetic composition of viral populations during the life cycle of Faba bean necrotic stunt virus (FBNSV) (genus Nanovirus), we demonstrate reproducible genetic changes during the transit of the virus within the body of the aphid vector. These changes do not fit the view that viruses simply traverse the bodies of their arthropod vectors and suggest more intimate interactions, calling into question the current understanding of circulative nonpropagative transmission.

  • Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model [Vaccines and Antiviral Agents]

  • Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated 145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 107 PFU of 145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 105 or 106 PFU of 145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. 145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with 145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P llt; 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 106 PFU (P llt; 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection.

    IMPORTANCE Previous attempts to develop successful immunization against cytomegalovirus have largely centered on subunit vaccination against virion proteins but have yielded disappointing results. The advent of bacterial artificial chromosome technologies has enabled engineering of recombinant cytomegaloviruses (CMVs) from which virus genome-encoded immune modulation genes have been deleted, toward the goal of developing a safe and potentially more efficacious live attenuated vaccine. Here we report the findings of studies of such a vaccine against congenital CMV infection based on a virus with a targeted deletion in gp145, a virus genome-encoded inhibitor of protein kinase R, using the guinea pig model of vertical CMV transmission. The deletion virus was attenuated for dissemination in immunocompromised guinea pigs but elicited ELISA and neutralizing responses. The vaccine conferred protection against maternal DNAemia and congenital transmission and resulted in reduced viral loads in newborn guinea pigs. These results provide support for future studies of attenuated CMV vaccines.

  • RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly [Structure and Assembly]

  • Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.

    IMPORTANCE Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

  • Expansion of Simian Immunodeficiency Virus (SIV)-Specific CD8 T Cell Lines from SIV-Naive Mauritian Cynomolgus Macaques for Adoptive Transfer [Cellular Response to Infection]

  • CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replication in vitro. We further cultured cell lines specific for four SIV-derived epitopes, Nef103nndash;111 RM9, Gag389nndash;394 GW9, Env338nndash;346 RF9, and Nef254nndash;262 LT9. These cell lines were up to 94.4% pure, as determined by major histocompatibility complex (MHC) tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks and trafficked to multiple extralymphoid tissues. However, these cells did not impact the acute-phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naive animals provide a unique model for exploring cellular immunity and the control of SIV infection and facilitate a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir.

    IMPORTANCE CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model.

  • Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity [Genetic Diversity and Evolution]

  • Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299 clones). Single-nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 x 10nndash;3 pplusmn; 0.4 x 10nndash;3; HCV, 2.1 x 10nndash;3 pplusmn; 0.5 x 10nndash;3) (P = 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 pplusmn; 0.6667 and 2.183 pplusmn; 0.9048, respectively) (P = 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 pplusmn; 0.02 and 0.83 pplusmn; 0.12, respectively) (P = 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection is not responsible for the higher worldwide genetic diversity observed in HCV.

    IMPORTANCE HCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection is responsible for the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in infected individuals. Our results indicate that HIV-1 and HCV protease quasispecies have very similar genetic diversity and comparable rugged enzymatic activity landscapes. Therapy for HCV has expanded, with new therapeutic agents such as the direct-acting antivirals (DAAs). DAAs, which target HCV NS3 protease and other virus proteins, have improved cure rates. However, major questions remain to be elucidated regarding the virologic correlates of HCV eradication. The findings shown here may help our understanding of the different therapeutic responses observed during chronic HCV infection.

  • Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation [Structure and Assembly]

  • During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ~12% to ~53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.

    IMPORTANCE Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.

  • HIV Vpu Interferes with NF-{kappa}B Activity but Not with Interferon Regulatory Factor 3 [Cellular Response to Infection]

  • The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu-mediated degradation of tetherin allows efficient particle release and hampers the activation of the NF-B pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However, the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the interferon regulatory factor 3 (IRF3) has been subject of conflicting reports. We therefore systematically investigated the expression of IRF3 in primary CD4+ T cells and macrophages infected with HIV at different time points. In addition, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF-B and the IRF3 pathways. We report here that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3-dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu downmodulated NF-B-dependent transcription. Mutation of two serines (positions 52 and 56) involved in the binding of NL4.3 Vpu to the bbeta;TrCP ubiquitin ligase abolishes its ability to inhibit NF-B activity. Taken together, these results suggest that HIV Vpu regulates antiviral innate response in primary human cells by acting specifically on the NF-B pathway.

    IMPORTANCE HIV Vpu plays a pivotal role in enhancing HIV infection by counteraction of Tetherin. However, Vpu also regulates host response to HIV infection by hampering the type 1 interferon response. The molecular mechanism by which Vpu inhibits the interferon response is still controversial. Here we report that Vpu affects interferon expression by inhibiting NF-B activity without affecting IRF3 levels or activity. These data suggest that Vpu facilitates HIV infection by regulating NF-B transcription to levels sufficient for viral transcription while limiting cellular responses to infection.

  • Replication of Tomato Yellow Leaf Curl Virus in Its Whitefly Vector, Bemisia tabaci [Pathogenesis and Immunity]

  • Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted exclusively by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial, and thus far, the virus has been considered to be nonpropagative. Following 8 h of acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (ggt;2-fold) during the first few days and then continuously decreased, as measured by the amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on whiteflies, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching of whiteflies between pesticide, heat stress, and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector.

    IMPORTANCE Plant viruses in agricultural crops are of great concern worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within these vectors. Such replication is important, as it contributes to the virus's spread and can impact vector biology. Tomato yellow leaf curl virus (TYLCV) is a devastating begomovirus that infects tomatoes. It is persistently transmitted by the whitefly Bemisia tabaci but is believed not to replicate in the insect. To demonstrate that TYLCV is, in fact, propagative (i.e., it replicates in its insect host), we hypothesized that insect defenses play a role in suppressing virus replication. We thus exposed whitefly to pesticide and heat stress conditions to manipulate its physiology, and we showed that under such conditions, the virus is able to replicate and significantly influence the insect's fecundity.

  • The Telomerase Inhibitor MST-312 Interferes with Multiple Steps in the Herpes Simplex Virus Life Cycle [Virus-Cell Interactions]

  • The life cycle of herpes simplex virus (HSV) has the potential to be further manipulated to yield novel, more effective therapeutic treatments. Recent research has demonstrated that HSV-1 can increase telomerase activity and that expression of the catalytic component of telomerase, telomerase reverse transcriptase (TERT), alters sensitivity to HSV-dependent apoptosis. Telomerase is a cellular enzyme that synthesizes nucleotide repeats at the ends of chromosomes (telomeres), which prevents shortening of the 3' ends of DNA with each cell division. Once telomeres reach a critical length, cells undergo senescence and apoptosis. Here, we used a cell-permeable, reversible inhibitor of the telomerase enzyme, MST-312, to investigate telomerase activity during HSV infection. Human mammary epithelial cells immortalized through TERT expression and human carcinoma HEp-2 cells were infected with the KOS1.1 strain of HSV-1 in the presence of MST-312. MST-312 treatment reduced the number of cells displaying a cytopathic effect and the accumulation of immediate early and late viral proteins. Moreover, the presence of 20 mmu;M to 100 mmu;M MST-312 during infection led to a 2.5- to 5.5-log10 decrease in viral titers. MST-312 also inhibited the replication of HSV-2 and a recent clinical isolate of HSV-1. Additionally, we determined that MST-312 has the largest impact on viral events that take place prior to 5 h postinfection (hpi). Furthermore, MST-312 treatment inhibited virus replication, as measured by adsorption assays and quantification of genome replication. Together, these findings demonstrate that MST-312 interferes with the HSV life cycle. Further investigation into the mechanism for MST-312 is warranted and may provide novel targets for HSV therapies.

    IMPORTANCE Herpes simplex virus (HSV) infections can lead to cold sores, blindness, and brain damage. Identification of host factors that are important for the virus life cycle may provide novel targets for HSV antivirals. One such factor, telomerase, is the cellular enzyme that synthesizes DNA repeats at the ends of chromosomes during replication to prevent DNA shortening. In this study, we investigate role of telomerase in HSV infection. The data demonstrate that the telomerase inhibitor MST-312 suppressed HSV replication at multiple steps of viral infection.

  • Simian Varicella Virus Is Present in Macrophages, Dendritic Cells, and T Cells in Lymph Nodes of Rhesus Macaques after Experimental Reactivation [Pathogenesis and Immunity]

  • Like varicella-zoster virus (VZV), simian varicella virus (SVV) reactivates to produce zoster. In the present study, 5 rhesus macaques were inoculated intrabronchially with SVV, and 5 months later, 4 monkeys were immunosuppressed; 1 monkey was not immunosuppressed but was subjected to the stress of transportation. In 4 monkeys, a zoster rash developed 7 to 12 weeks after immunosuppression, and a rash also developed in the monkey that was not immunosuppressed. Analysis at 24 to 48 h after zoster revealed SVV antigen in the lung alveolar wall, in ganglionic neurons and nonneuronal cells, and in skin and in lymph nodes. In skin, SVV was found primarily in sweat glands. In lymph nodes, the SVV antigen colocalized mostly with macrophages, dendritic cells, and, to a lesser extent, T cells. The presence of SVV in lymph nodes, as verified by quantitative PCR detection of SVV DNA, might reflect the sequestration of virus by macrophages and dendritic cells in lymph nodes or the presentation of viral antigens to T cells to initiate an immune response against SVV, or both.

    IMPORTANCE VZV causes varicella (chickenpox), becomes latent in ganglia, and reactivates to produce zoster and multiple other serious neurological disorders. SVV in nonhuman primates has proved to be a useful model in which the pathogenesis of the virus parallels the pathogenesis of VZV in humans. Here, we show that SVV antigens are present in sweat glands in skin and in macrophages and dendritic cells in lymph nodes after SVV reactivation in monkeys, raising the possibility that macrophages and dendritic cells in lymph nodes serve as antigen-presenting cells to activate T cell responses against SVV after reactivation.

  • Efficacy of the Herpes Simplex Virus 2 (HSV-2) Glycoprotein D/AS04 Vaccine against Genital HSV-2 and HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus Model [Vaccines and Antiviral Agents]

  • Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection.

    IMPORTANCE In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK HSV-2 vaccine Simplirix (gD/AS04) to protect humans against HSV-2 and the surprising finding that the vaccine protected against HSV-1 genital herpes instead. In this study, we report that gD/AS04 has higher efficacy against HSV-1 compared to HSV-2 genital herpes in the novel DMPA-synchronized cotton rat model of HSV-1 and HSV-2 infection. The findings help explain the results of the Simplirix trial.

  • Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication [Virus-Cell Interactions]

  • It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment.

    IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV.

  • Modulation of Glycosaminoglycans Affects PrPSc Metabolism but Does Not Block PrPSc Uptake [Prions]

  • Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrPSc. Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrPSc uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrPSc formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrPSc formation independent of the prion strain.

    IMPORTANCE Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.

  • Transcriptional Profiling of the Immune Response to Marburg Virus Infection [Pathogenesis and Immunity]

  • Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy.

    IMPORTANCE Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernible stages of response to infection that correlate with presymptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected, our results suggest that the innate immune response may contribute to overall pathogenesis.

  • Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques [Pathogenesis and Immunity]

  • Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (ggt;107 PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model.

    IMPORTANCE In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

  • Type I Interferon Released by Myeloid Dendritic Cells Reversibly Impairs Cytomegalovirus Replication by Inhibiting Immediate Early Gene Expression [Virus-Cell Interactions]

  • Cytomegalovirus (CMV) is a ubiquitous beta-herpesvirus whose reactivation from latency is a major cause of morbidity and mortality in immunocompromised hosts. Mouse CMV (MCMV) is a well-established model virus to study virus-host interactions. We showed in this study that the CD8-independent antiviral function of myeloid dendritic cells (mDC) is biologically relevant for the inhibition of MCMV replication in vivo and in vitro. In vivo ablation of CD11c+ DC resulted in higher viral titers and increased susceptibility to MCMV infection in the first 3 days postinfection. We developed in vitro coculture systems in which we cocultivated MCMV-infected endothelial cells or fibroblasts with T cell subsets and/or dendritic cells. While CD8 T cells failed to control MCMV replication, bone marrow-derived mDC reduced viral titers by a factor of up to 10,000. Contact of mDC with the infected endothelial cells was crucial for their antiviral activity. Soluble factors secreted by the mDC blocked MCMV replication at the level of immediate early (IE) gene expression, yet the viral lytic cycle reinitiated once the mDC were removed from the cells. On the other hand, the mDC did not impair MCMV replication in cells deficient for the interferon (IFN) alpha/beta receptor (IFNAR), arguing that type I interferons were critical for viral control by mDC. In light of our recent observation that type I IFN is sufficient for the induction of latency immediately upon infection, our results imply that IFN secreted by mDC may play an important role in the establishment of CMV latency.

    IMPORTANCE Numerous studies have focused on the infection of DC with cytomegaloviruses and on the establishment of latency within them. However, almost all of these studies have relied on the infection of DC monocultures in vitro, whereas DC are just one among many cell types present in an infection site in vivo. To mimic this aspect of the in vivo situation, we cocultured DC with infected endothelial cells or fibroblasts. Our data suggest that direct contact with virus-infected endothelial cells activates CD11c+ DC, which leads to reversible suppression of MCMV replication at the level of IE gene expression by a mechanism that depends on type I IFN. The effect matches the formal definition of viral latency. Therefore, our data argue that the interplay of dendritic cells and infected neighboring cells might play an important role in the establishment of viral latency.

  • Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells [Pathogenesis and Immunity]

  • Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-bbeta;) production. Our findings highlight the importance of IFN-bbeta; production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain.

    IMPORTANCE Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-bbeta; by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion.

  • A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1 [Pathogenesis and Immunity]

  • Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4+ (iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A+/NKG2Anndash; 3DL1+/3DL1nndash; (NKG2A+/nndash; 3DL1+/nndash;) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A+ 3DL1+ NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a+ IFN-+ CCL4+, CD107a+ IFN-+, total CD107a+, and total IFN-+ functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses.

    IMPORTANCE HIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A+ than NKG2Anndash; NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A+ and NKG2Anndash; NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.

  • Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013 [Genetic Diversity and Evolution]

  • The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like ("avian-like") genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level.

    IMPORTANCE The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like ("avian-like") lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

  • Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists [Virus-Cell Interactions]

  • Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

    IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

  • Transmission of H7N9 Influenza Viruses with a Polymorphism at PB2 Residue 627 in Chickens and Ferrets [Pathogenesis and Immunity]

  • Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 mmu;m. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intrahost" and "interhost" adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species.

    IMPORTANCE We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics.

  • Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1 [Pathogenesis and Immunity]

  • Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response.

    IMPORTANCE Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

  • Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis.

    IMPORTANCE This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.

  • Redundant Function of Plasmacytoid and Conventional Dendritic Cells Is Required To Survive a Natural Virus Infection [Pathogenesis and Immunity]

  • Viruses that spread systemically from a peripheral site of infection cause morbidity and mortality in the human population. Innate myeloid cells, including monocytes, macrophages, monocyte-derived dendritic cells (mo-DC), and dendritic cells (DC), respond early during viral infection to control viral replication, reducing virus spread from the peripheral site. Ectromelia virus (ECTV), an orthopoxvirus that naturally infects the mouse, spreads systemically from the peripheral site of infection and results in death of susceptible mice. While phagocytic cells have a requisite role in the response to ECTV, the requirement for individual myeloid cell populations during acute immune responses to peripheral viral infection is unclear. In this study, a variety of myeloid-specific depletion methods were used to dissect the roles of individual myeloid cell subsets in the survival of ECTV infection. We showed that DC are the primary producers of type I interferons (T1-IFN), requisite cytokines for survival, following ECTV infection. DC, but not macrophages, monocytes, or granulocytes, were required for control of the virus and survival of mice following ECTV infection. Depletion of either plasmacytoid DC (pDC) alone or the lymphoid-resident DC subset (CD8aalpha;+ DC) alone did not confer lethal susceptibility to ECTV. However, the function of at least one of the pDC or CD8aalpha;+ DC subsets is required for survival of ECTV infection, as mice depleted of both populations were susceptible to ECTV challenge. The presence of at least one of these DC subsets is sufficient for cytokine production that reduces ECTV replication and virus spread, facilitating survival following infection.

    IMPORTANCE Prior to the eradication of variola virus, the orthopoxvirus that causes smallpox, one-third of infected people succumbed to the disease. Following successful eradication of smallpox, vaccination rates with the smallpox vaccine have significantly dropped. There is now an increasing incidence of zoonotic orthopoxvirus infections for which there are no effective treatments. Moreover, the safety of the smallpox vaccine is of great concern, as complications may arise, resulting in morbidity. Like many viruses that cause significant human diseases, orthopoxviruses spread from a peripheral site of infection to become systemic. This study elucidates the early requirement for innate immune cells in controlling a peripheral infection with ECTV, the causative agent of mousepox. We report that there is redundancy in the function of two innate immune cell subsets in controlling virus spread early during infection. The viral control mediated by these cell subsets presents a potential target for therapies and rational vaccine design.

  • Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification [Genetic Diversity and Evolution]

  • Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it is not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus, rhtrs1, improved replication of a recombinant poxvirus (VVEK+RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations that improve virus replication with only a single copy of rhtrs1 to evolve, we passaged rhtrs1-amplified viruses in semipermissive primate cells. After passage, we isolated two viruses that contained only a single copy of rhtrs1 yet replicated as well as the amplified virus. Surprisingly, rhtrs1 was not mutated in these viruses; instead, we identified mutations in two vaccinia virus (VACV) genes, A24R and A35R, either of which was sufficient to improve VVEK+RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the antipoxviral drug isatin-bbeta;-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a "molecular foothold," broadly improving replication to facilitate rapid adaptation, while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification.

    IMPORTANCE Understanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of rhtrs1, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of rhtrs1-amplified viruses, there arose in two vaccinia virus genes mutations that improved viral replication without requiring rhtrs1 amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host.

  • Protection from Severe Influenza Virus Infections in Mice Carrying the Mx1 Influenza Virus Resistance Gene Strongly Depends on Genetic Background [Pathogenesis and Immunity]

  • Influenza virus infections represent a serious threat to human health. Both extrinsic and intrinsic factors determine the severity of influenza. The MX dynamin-like GTPase 1 (Mx1) gene has been shown to confer strong resistance to influenza A virus infections in mice. Most laboratory mouse strains, including C57BL/6J, carry nonsense or deletion mutations in Mx1 and thus a nonfunctional allele, whereas wild-derived mouse strains carry a wild-type Mx1 allele. Congenic C57BL/6J (B6-Mx1r/r) mice expressing a wild-type allele from the A2G mouse strain are highly resistant to influenza A virus infections, to both mono- and polybasic subtypes. Furthermore, in genetic mapping studies, Mx1 was identified as the major locus of resistance to influenza virus infections. Here, we investigated whether the Mx1 protective function is influenced by the genetic background. For this, we generated a congenic mouse strain carrying the A2G wild-type Mx1 resistance allele on a DBA/2J background (D2-Mx1r/r). Most remarkably, congenic D2-Mx1r/r mice expressing a functional Mx1 wild-type allele are still highly susceptible to H1N1 virus. However, pretreatment of D2-Mx1r/r mice with alpha interferon protected them from lethal infections. Our results showed, for the first time, that the presence of an Mx1 wild-type allele from A2G as such does not fully protect mice from lethal influenza A virus infections. These observations are also highly relevant for susceptibility to influenza virus infections in humans.

    IMPORTANCE Influenza A virus represents a major health threat to humans. Seasonal influenza epidemics cause high economic loss, morbidity, and deaths each year. Genetic factors of the host strongly influence susceptibility and resistance to virus infections. The Mx1 (MX dynamin-like GTPase 1) gene has been described as a major resistance gene in mice and humans. Most inbred laboratory mouse strains are deficient in Mx1, but congenic B6-Mx1r/r mice that carry the wild-type Mx1 gene from the A2G mouse strain are highly resistant. Here, we show that, very unexpectedly, congenic D2-Mx1r/r mice carrying the wild-type Mx1 gene from the A2G strain are not fully protected against lethal influenza virus infections. These observations demonstrate that the genetic background is very important for the protective function of the Mx1 resistance gene. Our results are also highly relevant for understanding genetic susceptibility to influenza virus infections in humans.

  • Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation [Structure and Assembly]

  • The herpesviral terminase complex is part of the intricate machinery that delivers a single viral genome into empty preformed capsids (encapsidation). The varicella-zoster virus (VZV) terminase components (pORF25, pORF30, and pORF45/42) have not been studied as extensively as those of herpes simplex virus 1 and human cytomegalovirus (HCMV). In this study, VZV bacterial artificial chromosomes (BACs) were generated with small (30S), medium (30M), and large (30L) ORF30 internal deletions. In addition, we isolated recombinant viruses with specific alanine substitutions in the putative zinc finger motif (30-ZF3A) or in a conserved region (region IX) with predicted structural similarity to the human topoisomerase I core subdomains I and II (30-IXAla, 30-620A, and 30-622A). Recombinant viruses replicated in an ORF30-complementing cell line (ARPE30) but failed to replicate in noncomplementing ARPE19 and MeWo cells. Transmission electron microscopy of 30-IXAla-, 30-620A-, and 30-622A-infected ARPE19 cells revealed only empty VZV capsids. Southern analysis showed that cells infected with parental VZV (VZVLUC) or a repaired virus (30R) contained DNA termini, whereas cells infected with 30L, 30-IXAla, 30-620A, or 30-622A contained little or no processed viral DNA. These results demonstrated that pORF30, specifically amino acids 619 to 624 (region IX), was required for DNA encapsidation. A luciferase-based assay was employed to assess potential intermolecular complementation between the zinc finger domain and conserved region IX. Complementation between 30-ZF3A and 30-IXAla provided evidence that distinct pORF30 domains can function independently. The results suggest that pORF30 may exist as a multimer or participate in higher-order assemblies during viral DNA encapsidation.

    IMPORTANCE Antivirals with novel mechanisms of action are sought as additional therapeutic options to treat human herpesvirus infections. Proteins involved in the viral DNA encapsidation process have become promising antiviral targets. For example, letermovir is a small-molecule drug targeting HCMV terminase that is currently in phase III clinical trials. It is important to define the structural and functional characteristics of proteins that make up viral terminase complexes to identify or design additional terminase-specific compounds. The VZV ORF30 mutants described in this study represent the first VZV terminase mutants reported to date. Targeted mutations confirmed the importance of a conserved zinc finger domain found in all herpesvirus ORF30 terminase homologs but also identified a novel, highly conserved region (region IX) essential for terminase function. Homology modeling suggested that the structure of region IX is present in all human herpesviruses and thus represents a potential structurally conserved antiviral target.

  • The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication [Virus-Cell Interactions]

  • Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription.

    IMPORTANCE Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member of the Microrchidia family that is associated with the nuclear matrix, during virus infection. NXP2/MORC3 associates with the viral ribonucleoproteins in infected cells. Downregulation of NXP2/MORC3 reduced virus titers and accumulations of viral genomic RNA and mRNAs. Silencing of NXP2/MORC3 in an influenza virus CAT minireplicon system diminished CAT protein and cat mRNA levels but not genomic RNA levels. We propose that NXP2/MORC3 plays a role in influenza virus transcription.

  • Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation [Virus-Cell Interactions]

  • Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.

    IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

  • Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate [Prions]

  • Thirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds.

    IMPORTANCE This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

  • Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action [Vaccines and Antiviral Agents]

  • Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface.

    IMPORTANCE In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.

  • Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation [Genome Replication and Regulation of Viral Gene Expression]

  • Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA.

    IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.

  • Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry [Virus-Cell Interactions]

  • The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ~9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry.

    IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.

  • Human Cytotoxic T Lymphocyte-Mediated Acute Liver Failure and Rescue by Immunoglobulin in Human Hepatocyte Transplant TK-NOG Mice [Pathogenesis and Immunity]

  • Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B.

    IMPORTANCE Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by human HBV-specific CTLs. In this model, HBV-infected human hepatocytes were transplanted into severely immunodeficient NOG mice in order to reconstruct elements of the human immune system. Using this model, we found that CTL-associated antigen-4 immunoglobulin was able to suppress damage to HBV-infected hepatocytes, suggesting an approach to treatment. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B.

  • Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1 [Genome Replication and Regulation of Viral Gene Expression]

  • Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1' and D1', in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI.

    IMPORTANCE Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus.

  • Latent Expression of the Epstein-Barr Virus (EBV)-Encoded Major Histocompatibility Complex Class I TAP Inhibitor, BNLF2a, in EBV-Positive Gastric Carcinomas [Transformation and Oncogenesis]

  • The Epstein-Barr virus (EBV) BNLF2a gene product provides immune evasion properties to infected cells through inhibition of transporter associated with antigen processing (TAP)-mediated transport of antigen peptides. Although BNLF2a is considered to be a lytic gene, we demonstrate that it is expressed in nearly half of the EBV-associated gastric carcinomas analyzed. Further, we show that BNLF2a expression is dissociated from lytic gene expression. BNLF2a is therefore expressed in this latency setting, potentially helping protect the infected tumor cells from immunosurveillance.

  • 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1 [Virus-Cell Interactions]

  • 2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

  • The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

  • Determinants of Human CD134 Essential for Entry of Human Herpesvirus 6B [Cellular Response to Infection]

  • We identified two key amino acid residues within human CD134 (hCD134) that are required for its interaction with human herpesvirus 6B (HHV-6B) and for HHV-6B entry into cells. One of the residues (K79) allows access of the HHV-6B ligand to hCD134. Murine CD134 (mCD134) functioned as an HHV-6B receptor when these two amino acid residues were replaced with homologous human residues. This study identifies both the HHV-6B receptor-ligand interaction and the species-specific determinants of hCD134 essential for HHV-6B entry.

  • Puzzling Origins of the Ebola Outbreak in the Democratic Republic of the Congo, 2014 [Letters to the Editor]

  • Correction for Bitzegeio et al., Adaptation to the Interferon-Induced Antiviral State by Human and Simian Immunodeficiency Viruses [Author Correction]

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    JVI Accepts: Articles Published Ahead of Print

  • Down-regulation of toll-like receptor 9 expression by beta human papillomavirus type 38 and implications for cell cycle control [PublishAheadOfPrint]

  • Innate immunity is the first line of host defence against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus (HPV) type 38 E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor TLR9 in human foreskin keratinocytes (HFK), a key mediator of the anti-viral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA down regulation by promoting accumulation of Np73aalpha;, an antagonist of p53 and p73. Inhibition of Np73aalpha; expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of Np73aalpha; in this event. Chromatin immunoprecipitation experiments showed that Np73aalpha; is part of a negative transcriptional regulatory complex with IB kinase beta (IKKbbeta;) that binds to a NF-B responsive element within the TLR9 promoter. In addition, the polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex leading to histone 3 tri-methylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21WAF1 and p27Kip1, decreased CDK2-associated kinase activity and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity can down-regulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation.

    IMPORTANCE The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune-system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV type 38 down-regulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated down-regulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

  • Mapping enterovirus A71 antigenic determinants from viral evolution [PublishAheadOfPrint]

  • Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effect of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability, and that surveillance studies should look out for these mutants that could compromise vaccine efficacy.

    IMPORTANCE Emerging and re-emerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants, and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.

  • STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-associated Herpesvirus [PublishAheadOfPrint]

  • Lytic activation of Kaposi's Sarcoma-associated Herpesvirus (KSHV) from latency is a critical contributor to pathogenesis and progression of KSHV-mediated disease. Development of targeted treatment strategies and improvement of lytic phase-directed oncolytic therapies therefore hinge on gaining a better understanding of latency-to-lytic transition. A key observation in that regard, also common to other herpesviruses, is the partial permissiveness of latently-infected cells to lytic cycle inducing agents. Here, we address the molecular basis for why only some KSHV-infected cells respond to lytic stimuli. Since cellular Signal Transducer and Activator of Transcription 3 (STAT3) is overactive in KSHV-associated cancers, KSHV activates STAT3, and STAT3 has been found to regulate lytic activation of Epstein-Barr virus (EBV)-infected cells, we asked if STAT3 contributes similarly to the life cycle of KSHV. We found that high levels of STAT3 correlate with the refractory state at the single cell level under conditions of both spontaneous and induced lytic activation; importantly, STAT3 also regulates lytic susceptibility. Further, knockdown of STAT3 suppresses the cellular transcriptional co-repressor Kruuuml;ppel-associated box domain-associated protein 1 (KAP1), and suppression of KAP1 activates lytic genes including the viral lytic switch RTA, thereby linking STAT3 via KAP1 to regulation of the balance between lytic and latent cells. These findings, taken together with those from EBV-infected, and more recently HSV-1-infected cells, cement the contribution of host STAT3 to persistence of herpesviruses, and simultaneously reveal an important lead to devise strategies to improve lytic phase-directed therapies for herpesviruses.

    IMPORTANCE Lytic activation of the cancer-causing Kaposi's Sarcoma-associated Herpesvirus (KSHV) is vital to its life cycle and causation of disease. Like other herpesviruses, however, a substantial fraction of latently-infected cells are resistant to lytic-inducing stimuli. Investigating the molecular basis for this refractory state is essential for understanding how the virus persists, how it causes disease, and to guide efforts to improve treatment of KSHV-mediated diseases. We find that like two other herpesviruses, EBV and HSV-1, KSHV exploits the cellular transcription factor STAT3 to regulate susceptibility of latently-infected cells to lytic triggers. These findings highlight a common STAT3-centered strategy used by herpesviruses to maintain persistence in their host, while also revealing a key molecule to pursue while devising methods to improve herpesvirus lytic phase-directed therapies.

  • Immune protection against virus challenge in aging mice is not affected by latent herpesviral infections [PublishAheadOfPrint]

  • Latent herpesvirus infections alter the immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse CMV, HSV-1 and/or MHV-68 with Influenza, WNV or VSV. We observed no increase in mortality or weight-loss over herpesvirus-negative counterparts and a relative, but no absolute reduction in CD8 responses against acute infections. Therefore, herpesviruses do not appear to increase susceptibility to emerging infections in aging.

  • The ns12.9 accessory protein of human coronavirus OC43 is a viroporin involved in virion morphogenesis and pathogenesis [PublishAheadOfPrint]

  • An accessory gene between the S and E gene loci is contained in all coronaviruses (CoVs), and its function has been studied in some coronaviruses. This gene locus in human coronavirus OC43 (HCoV-OC43) encodes the ns12.9 accessory protein; however, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein (HCoV-OC43-ns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-ns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo. Intriguingly, exogenous ns12.9 and heterologous viroporins with ion channel activity could compensate the production of HCoV-OC43-ns12.9, indicating that the ion channel activity of ns12.9 plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication revealed that ns12.9 protein had no measurable effect on virus entry, subgenomic messenger RNA (sgmRNA) synthesis and protein expression. Further characterization revealed that HCoV-OC43-ns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus (HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response and virulence in HCoV-OC43-ns12.9-infected mice were observed compared with HCoV-OC43-WT-infected mice. Taken together, our results demonstrated that the ns12.9 accessory protein functions as a viroporin, and is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection.

    IMPORTANCE HCoV-OC43 was isolated in the 1960s and is a major agent of the common cold. The functions of HCoV-OC43 structural proteins have been well studied, but few studies have focused on its accessory proteins. In the present study, we demonstrated that the ns12.9 protein is a newly recognized viroporin, and the ns12.9 gene knockout virus (HCoV-OC43-ns12.9) presents a growth defect in vitro and in vivo. We identified the important functions of the ns12.9 viroporin in virion morphogenesis during HCoV-OC43 infection. Furthermore, mice infected with HCoV-OC43-ns12.9 exhibited reduced inflammation and virulence accompanied by a lower titer in the brain compared with wild-type infected mice, suggesting the ns12.9 viroporin influences virus pathogenesis. Therefore, our findings revealed that the ns12.9 viroporin facilitates the virion morphogenesis to enhance viral production, and these results provided a deeper understanding of HCoV-OC43 pathogenesis.

  • HSV capsid/organelle association in the absence of the large tegument protein UL36p [PublishAheadOfPrint]

  • UL36p (VP1/2) is the largest protein encoded by HSV-1 and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens, and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that, while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid/membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin section electron microscopy and correlative light and electron microscopy. We conclude that capsid/membrane binding can occur in the absence of UL36p, and propose that this association may precede the events of UL36p-driven envelopment.

    IMPORTANCE Membrane-association and envelopment of the HSV capsid is essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins, however the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment.

  • Structural basis of the inhibition of STAT1 activity by Sendai virus C protein [PublishAheadOfPrint]

  • Sendai virus C protein inhibits the signal transduction pathways of interferon (IFN)-aalpha;/bbeta; and IFN- by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3, amino acids 99nndash;204) at a resolution of 2.0 AAring;. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an anti-parallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anti-cooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr701 by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated (pY)-STAT1 with Y3 associated with the -activated sequence probably as high molecular weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN- by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-aalpha;/bbeta;.

    IMPORTANCE Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon-aalpha;/bbeta; and interferon- by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr701 residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr701, further inducing high molecular weight complex formation and inhibition of transcription by interferon-. We also discuss the possible mechanism of inhibition of the interferon-aalpha;/bbeta; pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

  • Molecular determinants of virulence and stability of a reporter-expressing H5N1 influenza A virus [PublishAheadOfPrint]

  • We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from A/Puerto Rico/8/1934 (H1N1) virus (Venus-H5N1 virus), became more lethal to mice and the reporter gene was more stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of WT-Venus-H5N1 virus in mice, and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus.

    IMPORTANCE The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virus-related live imaging studies in vitro and in vivo.

  • Epstein-Barr viruses deficient in EBER RNAs give higher LMP2 RNA expression in lymphoblastoid cell lines and efficiently establish persistent infection in humanized mice [PublishAheadOfPrint]

  • Functions of EBER RNAs were tested in lymphoblastoid cell lines containing EBER mutants of Epstein-Barr Virus (EBV). Binding of EBER1 to RPL22 was confirmed. Deletion of EBER1 or EBER2 correlated with increased cytoplasmic EBV LMP2 RNA and with small effects on specific cellular miRNA levels but protein levels of LMP1 and LMP2A were not affected. Wild type and EBER deletion EBV had approximately equal ability to infect immunodeficient mice reconstituted with a human haematopoietic system.

  • Release of VSVG-pseudotyped Lentivirus from the Host Cell is impaired upon Low-density Lipoprotein Receptor Overexpression [PublishAheadOfPrint]

  • Production of a VSVG-pseudotyped lentiviral expression vector in HEK293 cells decreased on overexpression of LDLR but not of ICAM1 or TfR1. RT-qPCR revealed a reduction in vector RNA as a function of LDLR expression. Decreased syncytia formation suggested diminished surface expression of VSVG. Intracellular VSVG granules colocalized with LDLR, ERGIC53, LAMP2, and vimentin, but neither with GM130 nor calnexin suggesting that VSVG interacts with LDLR within the ERGIC resulting in re-routing into the aggresome/autophagosome pathway.

  • The genome of a tortoise herpesvirus (testudinid herpesvirus 3) has a novel structure and contains a large region that is not required for replication in vitro or virulence in vivo [PublishAheadOfPrint]

  • Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step towards characterizing TeHV-3 and developing prophylactic methods against it.

    IMPORTANCE Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome, and used this information to take steps towards developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are: (i) TeHV-3 has a novel genome structure; (ii) its closest relative is a turtle herpesvirus; (iii) it contains interleukin-10 and semaphorin genes, the first time these have been reported in an alphaherpesvirus; (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo; and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

  • Influences on the design and purification of soluble, recombinant native-like HIV-1 envelope glycoprotein trimers [PublishAheadOfPrint]

  • We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and fully native-like. They also have a native-like (i.e., abundant) oligomannose glycan composition, and display multiple bNAb epitopes while occluding those for non-neutralizing antibodies. In contrast, uncleaved, Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker between the gp120 and gp41ECTO subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ~20-30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.

    IMPORTANCE Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surface of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.

  • Long-term effect of serial infections with H13 and H16 low pathogenic avian influenza viruses in black-headed gulls [PublishAheadOfPrint]

  • Infections of domestic and wild birds with low pathogenic avian influenza viruses (LPAIV) have been associated with protective immunity to subsequent infection. However, the degree and duration of immunity in wild birds from previous LPAIV infectionmmdash;by the same or a different subtypemmdash;are poorly understood. Therefore, we inoculated H13N2 (A/Black-headed gull/Netherlands/7/2009) and H16N3 (A/Black-headed gull/Netherlands/26/2009) LPAIVs into black-headed gulls (Chroicocephalus ridibundus)mmdash;their natural host speciesmmdash;and measured the long-term immune response and protection against one or two re-infections over a period of more than one year. This is the typical interval between LPAIV epizootics in wild birds. Re-infection with the same virus resulted in progressively less virus excretion, with complete abrogation of virus excretion after two infections for H13 but not H16. However, re-infection with the other virus affected neither level nor duration of virus excretion. Virus excretion by immunologically naiiuml;ve birds did not differ in total level of excreted H13 or H16 virus between first- and second-year birds, but duration of H13 excretion was shorter for second-year birds. Furthermore, serum antibody levels did not correlate with protection against LPAIV infection. LPAIV-infected gulls showed no clinical signs of disease. These results imply that the epidemiological cycles of H13 and H16 in black-headed gulls are relatively independent from each other and depend mainly on infection of first-year birds.

    IMPORTANCE Low pathogenic avian influenza viruses (LPAIV) circulate mainly in wild waterbirds, but are occasionally transmitted to other species including humans, where they cause subclinical to fatal disease. To date, the effect of LPAIV-specific immunity on the epidemiology of LPAIV in wild birds is poorly understood. In this study, we investigated the effect of H13 and H16 LPAIV infection in black-headed gulls on susceptibility and virus excretion of subsequent infection with the same or the other virus within the same breeding season and between breeding seasons. These are the only two LPAIV hemagglutinin subtypes predominating in this species. The findings suggest H13 and H16 LPAIV cycles in black-headed gull populations are independent of each other, indicate the importance of first-year birds in LPAIV epidemiology and emphasize the need for alternatives to AIV-specific serum antibodies as evidence of past LPAIV infection and correlate of protection against LPAIV infection in wild birds.

  • HuR displaces PTB to facilitate La binding to the 3' UTR and enhances HCV replication [PublishAheadOfPrint]

  • HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here we report a novel role for HuR, as a regulator of proteins assembling at the 3rrsquo; untranslated region of viral RNA in the context of Hepatitis C virus infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B) and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3rrsquo; UTR of the viral RNA affects the interaction of two host proteins La and PTB at this site. HuR interacts with La and facilitates La binding to the 3rrsquo; UTR, enhancing La mediated circularization of the HCV genome and thus viral replication. In addition, it competes with the polypyrimidine tract binding protein (PTB) for association with the 3rrsquo; UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through interplay of host and viral proteins, orchestrated by HuR.

    IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3rrsquo; untranslated region (3rrsquo; UTR) of HCV RNA. At the 3rrsquo; UTR, it facilitates circularization of the viral genome through interaction with another host factor La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unravelled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

  • Viral small RNA analysis of Bombyx mori larval midgut during persistent and pathogenic cytoplasmic polyhedrosis virus infection [PublishAheadOfPrint]

  • Lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by employing deep sequencing technology for viral small RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented dsRNA genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20 nt vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot- and cold-spot profiles for each viral segment, are to a considerable degree overlapping between Dicer-2-related (19-21 nt) and Dicer-2-unrelated vsRNAs suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown ribonuclease to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.

    IMPORTANCE This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented dsRNA virus (CPV; Reoviridae) and highlights the importance of vsRNA analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20 nt vsRNAs, or based on the activity of an uncharacterized endoRNase that cleaves the viral RNA substrate at a degenerate motif. The analysis indicates also the existence of a degradation pathway that targets the (+)-strand of segment 10.

  • Preferential budding of vesicular stomatitis virus from the basolateral surface of polarized epithelial cells is not solely directed by matrix protein or glycoprotein [PublishAheadOfPrint]

  • VSV has been shown to bud basolaterally, and the matrix protein but not glycoprotein was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.

  • Th1/17 polarization of CD4 T cells supports HIV-1 DNA persistence during antiretroviral therapy [PublishAheadOfPrint]

  • The ability to persist long term in latently-infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggest that only small subsets of CD4 T cells with specific developmental and maturational profiles are capable to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1 infected cells. We found that CD4 T cells enriched for a Th1/Th17 polarization had elevated susceptibilities to HIV-1 infection in ex-vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/Th17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/Th17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy.

    IMPORTANCE Current antiretroviral therapy is very effective in suppressing active HIV-1 replication, but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long-term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/Th17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally ascribed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.

  • Differential PI3K-Akt-mTOR activation by Semliki Forest and chikungunya virus, dependent on nsP3 and connected to replication complex internalisation [PublishAheadOfPrint]

  • Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)nndash;Aktnndash;mammalian target of Rapamycin (mTOR) pathway, a crucial pro-survival signalling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV non-structural protein nsP3. Viruses with a deletion of this domain (SFV-50), but not other regions in nsP3, displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of nsP3-wildtype, but not -50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3Knndash;Aktnndash;mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalised in a process blocked by the PI3K inhibitor Wortmannin. Replication complex internalisation was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3, but not with SFV-50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3Knndash;Aktnndash;mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3Knndash;Aktnndash;mTOR activation and replication complex internalisation in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3Knndash;Aktnndash;mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalisation.

    Importance SFV and CHIKV are very similar in terms of molecular and cell biology, e.g. regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that SFV and CHIKV both activate the pro-survival PI3Knndash;Aktnndash;mTOR pathway in cells, but greatly differ in their capacity to do so: Akt is strongly and persistently activated by SFV infection, but only moderately by CHIKV. We mapped this activation capacity to a region in the non-structural protein 3 of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalised from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localisation may have implications for pathology.

  • Characterising the diverse mutational pathways associated with R5-tropic maraviroc resistance: HIV-1 that uses the drug-bound CCR5 coreceptor [PublishAheadOfPrint]

  • Entry-inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to pre-existing virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use drug-bound CCR5 coreceptor. To understand this R5-resistance pathway we analysed ggt;500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2 in which treatment experienced patients received maraviroc plus optimised background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis shows the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there is a notable lack of commonality in the specific sites conferring resistance confirming the unusual nature of R5-tropic resistance. We use coevolutionary and positive selection analysis to characterise the genotypic determinants of resistance and find: (1) complicated covariation networks indicating frequent coevolutionary/compensatory changes in the context of protein structure; (2) covarying sites under positive selection are enriched in resistant viruses; (3) CD4 binding sites form part of a unique covariation network independent of the V3 loop; (4) the covariation network formed between the V3 loop, and other regions of gp120 and gp41 intersects with sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context-dependent and thus inherently unpredictable.

    Importance The entry-inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug resistant virus is particularly interesting because it tends to be rare with lack of sensitivity usually associated with the presence of CXCR4-using virus (the main alternative coreceptor HIV-1 uses in addition to CD4). We analysed envelope sequences from HIV-1 obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We find that in these individuals, resistant viral populations form a distinct population that evolved once and were successful as a result of the drug pressure. Further evolutionary analysis places the complex network of inter-dependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

  • Tracking the fate of genetically distinct vesicular stomatitis virus matrix proteins highlights the role for late domains in assembly [PublishAheadOfPrint]

  • Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) to the large polymerase protein (L). To study assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of insertions in the M gene, a replication competent virus containing a fluorescent M, and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintain the same bullet-shape appearance as wild type VSV with a modest increase in particle length reflecting the increased genome size. Imaging of the released particles reveals a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the "late" domains in M, which serve to recruit the endosomal sorting complexes required for transport (ESCRT) machinery during budding. Mutations in late domains result in the accumulation of virions that fail to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M-eGFP and M-mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late domain mutant into the released virions. By contrast, intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.

    Importance Assembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein to the plasma membrane. The matrix protein contains a motif termed a "late domain" that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. Here we develop new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We use those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

  • SLAM family receptor homologs in New World monkey cytomegaloviruses [PublishAheadOfPrint]

  • Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signalling lymphocytic activation molecule (SLAM) family receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAM family (SLAMF) genes encoded by two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host co-evolution. The most recent acquisition led to S1 in SMCMV, which encodes a SLAMF6 homolog with an amino acid identity of 97% in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell-surface glycoprotein capable of binding to host SLAMF6. Furthermore, OMCMV encodes A33, a LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counter-receptors, and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found in both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signalling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural moulds for expanding their immunomodulatory repertoires.

    IMPORTANCE The way in which viruses shape their genomes, under the continual selective pressure exerted by the host immune system, is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune-cell receptors of the signalling lymphocyte activation molecule (SLAM) family during host-virus co-evolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties, including soluble forms and absence of typical SLAM signalling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution.

  • Modification of the asparagine-linked glycan density for the design of hepatitis B virus (HBV) virus-like particles with enhanced immunogenicity [PublishAheadOfPrint]

  • The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine, and are platforms for the delivery of foreign antigenic sequences. To investigate structurenndash;immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified VLPs exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wildtype (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced sites T116N and G130N were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamins, and sialylation. Antigenic fingerprints for the WT, hypo-, and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their binding ability to the different VLP glyco-analogues suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titres. The T116N VLPs induced earlier and longer lasting antibody responses compared to the hypoglycosylated and WT VLPs. The ability of non-native VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with the innate immune system illustrates pathways for the design of immunogens for superior preventive applications.

    IMPORTANCE The use of biochemically modified, non-native immunogens represent an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized demonstrating that they carry the same types of glycans as wildtype VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer lasting antibody immune responses compared to wildtype or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

  • Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly [PublishAheadOfPrint]

  • The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon-aalpha; therapy vary based on HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBVG2335A expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pgRNA was higher than that in cells transfected with an HBV genotype-A expression plasmid. By using co-transfection with an HBV genotype-D and 2.2DS-RNA plasmids, we found that reduction of pgRNA was observed in the cells even in low amount of the 2.2DS-RNA plasmids. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line, 1.3ES2, reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and PABP1, in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and response to interferon-aalpha; therapy between patients infected with different HBV genotypes.

    IMPORTANCE Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma, and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.

  • Phenotypic Correlates of HIV-1 Macrophage Tropism [PublishAheadOfPrint]

  • HIV-1 is typically CCR5-using (R5) and T cell-tropic (T-tropic), targeting memory CD4+ T cells throughout acute and chronic infection. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies, but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation.

    IMPORTANCE HIV-1 typically replicates in CD4+ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multi-step process to macrophage tropism.

  • Spatiotemporal reconstruction of the introduction of hepatitis C virus into Scotland and its subsequent regional transmission [PublishAheadOfPrint]

  • A more comprehensive understanding of hepatitis C virus (HCV) transmission dynamics could facilitate public health initiatives to reduce the prevalence of HCV in people who inject drugs. We aimed to determine how HCV sequences entered and spread throughout Scotland and to identify transmission hotspots. A Scottish dataset with embedded demographic data was created by sequencing the NS5B of 125 genotype (Gt) 1a and 166 Gt3a samples and analysed alongside sequences from public databases. Applying Bayesian inference methods, we reconstructed the global origin and local spatiotemporal dissemination of HCV in Scotland. Scottish sequences mainly formed discrete clusters interspersed between sequences from the rest of the world; the most recent common ancestors of these clusters dated to 1942-1952 (Gt1a) and 1926-1942 (Gt3a), coincident with global diversification and distribution. Extant Scottish sequences originated in Edinburgh (Gt1a) and Glasgow (Gt3a) in the 1970s but both genotypes spread from Glasgow to other regions. The dominant Gt1a strain differed between Edinburgh (cluster [C]2), Glasgow (C3) and Aberdeen (C4) whereas significant Gt3a strain-specificity only occurred in Aberdeen. Specific clusters initially formed separate transmission zones in Glasgow that subsequently overlapped occasioning city-wide co-circulation. Transmission hotspots were detected with 45% of samples from patients residing in just nine of Glasgow's 57 postcode districts. HCV was introduced into Scotland in the 1940s concomitant with its worldwide dispersal likely arising from global-scale historical events. Cluster-specific transmission hubs were identified in Glasgow, the key Scottish city implicated in HCV dissemination. This fine-scale spatiotemporal reconstruction improves understanding of HCV transmission dynamics in Scotland.

    IMPORTANCE HCV is a major health burden and the leading cause of hepatocellular carcinoma. Public health needle exchange and "treatment as prevention" strategies targeting HCV are designed to reduce prevalence of the virus in people who inject drugs (PWID), potentially mitigating the future burden of HCV-associated liver disease. Understanding HCV transmission dynamics could increase the effectiveness of such public health initiatives by identifying and targeting regions playing a central role in virus dispersal. In this study we examined HCV transmission in Scotland by analysing the genetic relatedness of strains from PWID alongside data inferring the year individuals became infected and residential information at a geographically finer-scale resolution than in previous studies. Clusters of Scotland-specific strains were identified with regional specificity and mapping the spread of HCV allowed the identification of key areas central to HCV transmission in Scotland. This research provides a basis for identifying HCV transmission hotspots.

  • PAMP Recognition of Hepatitis C Virus Transmitted/founder Variants by RIG-I is dependent on U-Core length [PublishAheadOfPrint]

  • Despite introduction of direct acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV is supported by a variety of viral immune evasion strategies but remarkably 20-30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV. However, the virus/host interactions regulating acute infection are unknown. Transmission of HCV involves one or few transmitted/founder (T/F) variants. In infected hepatocytes the retinoic acid inducible gene-I (RIG-I) protein recognizes 5rrsquo; triphosphate (5rrsquo; ppp) of the HCV RNA and a pathogen associated molecular pattern (PAMP) motif located within the 3rrsquo; untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can vary in length and complexity, suggesting PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected patients, we tested whether RIG-I recognition and innate immune activation correlates with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP, and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-to-chronic transition.

    IMPORTANCE Recognition of non-self molecular patterns such as viral nucleic acids is an essential step in triggering the immune response against virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein wherein RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I binding and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling sufficient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact transition from acute-to-chronic infection.

  • Remote activation of host cell DNA synthesis in uninfected cells signalled by infected cells in advance of virus transmission [PublishAheadOfPrint]

  • Viruses modulate cellular processes and metabolism in diverse ways but these are almost universally studied in the infected cell itself. Here we study spatial organisation of DNA synthesis during multi-round transmission of herpes simplex virus (HSV) using pulse-labelling with ethynyl-nucleotides and cycloaddition of azide-fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with current understanding during single step growth cycle, HSV suppresses host DNA synthesis and promotes virus DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover using pulse chase analysis we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis was then shut off and replaced with virus DNA synthesis. Using non-propagating viruses or conditioned medium we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as for mechanisms governing host DNA synthesis.

    Importance We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus induced paracrine effector. The field has had no conception that this process occurs and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrating the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multi-round transmission versus single-step growth curves, reveal a hitherto unknown processes in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signalling of other processes including transcription and protein synthesis.

  • The combination of the R263K and T66I resistance substitutions in HIV-1 integrase is incompatible with high level viral replication and the development of high-level drug resistance [PublishAheadOfPrint]

  • Background: The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and been reported in several treatment-experienced individuals receiving DTG as part of salvage therapy. R263K seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG- treated individuals.

    Methods: Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand-transfer and 3rrsquo; processing activities were measured biochemically.

    Results: The combination of R263K and T66I decreased HIV-1 infectivity, replicative capacity, and strand-transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL.

    Conclusions: These findings suggest that the presence of T66I will not compromise the activity of DTG and may also help to prevent the additional generation of R263K. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution.

    Importance The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus-1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution whereas R263K is a signature resistant substitution against dolutegravir. In order to determine how different combinations of resistance integrase mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand-transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir who harbor the T66I resistance substitution.

  • cGAS and STING knockout rescues virus infection of plasmid DNA-transfected cells [PublishAheadOfPrint]

  • It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid CRISPR/Cas9 system, we generated cGAS/STING knockout cells and show that these cells can as efficiently be infected after plasmid DNA transfection as non-transfected cells.

  • Characterisation of the interaction between the matrix protein of vesicular stomatitis virus and the immunoproteasome subunit LMP2. [PublishAheadOfPrint]

  • The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation and cellular pathogenesis. Using a yeast two hybrid system, M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by co-immunoprecipitation of M and LMP2 in VSV infected cells . The sites of interaction were characterized. A single mutation of M (I96A), which significantly impairs the interaction between M and LMP2, was identified. We also show that M binds preferentially to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket, where the protein substrate binds prior to cleavage, and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds LMP2 before its incorporation in the immunoproteasome. As the immunoproteasome promotes the generation of MHC class-I compatible peptides, a feature which favours the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism to escape the detection and elimination of infected cells by the immune system.

    IMPORTANCE The immunoproteasome promotes the generation of MHC class I compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome specific catalytic subunit. M protein preferentially binds LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds LMP2 before its incorporation in the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism to escape or limit the detection and elimination of infected cells by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

  • Bone marrow stromal antigen-2 (Bst2)/tetherin is induced in neurons by type I interferon and viral infection, but is dispensable for protection against neurotropic viral challenge [PublishAheadOfPrint]

  • In permissive mouse central nervous system (CNS) neurons, measles virus (MV) spreads in the absence of hallmark viral budding or neuronal death, with transmission occurring efficiently and exclusively via the synapse. MV infection also initiates a robust type I interferon (Ifn) response, resulting in the synthesis of a large number of genes, including bone marrow stromal antigen-2 (Bst2)/tetherin/CD317. Bst2 restricts the release of some enveloped viruses, but to date, its role in viral infection of neurons has not been assessed. Consequently, we investigated how Bst2 was induced, and what role it played in MV neuronal infection. The magnitude of induction of neuronal Bst2 RNA and protein following Ifn exposure and viral infection was notably higher than in similarly treated mouse embryo fibroblasts (MEFs). Bst2 synthesis was both Ifn- and Stat1-dependent. Although Bst2 prevented MV release from non-neuronal cells, its deletion had no effect on viral pathogenesis in MV-challenged mice. Our findings underscore cell type-specific differences in Bst2 function, and its impact on viral infection and pathogenesis.

    IMPORTANCE Viral infections of the central nervous system can lead to debilitating disease and death. Moreover, it is becoming increasingly clear that non-renewable cells, including most central nervous system neurons, combat neurotropic viral infections in fundamentally different ways than other rapidly dividing and renewable cell populations. Here we identify type I interferon signaling as a key inducer of a known anti-viral protein (Bst2) in neurons. Unexpectedly, this gene is dispensable for clearance of neurotropic viral infection despite its well-defined contribution to limiting the spread of enveloped viruses in proliferating cells. A deeper appreciation of the importance of cell-type heterogeneity in antiviral immunity will aid in the identification of unique therapeutic targets for life-threatening viral infections.

  • OLIGOMERIZATION OF MUMPS VIRUS PHOSPHOPROTEIN [PublishAheadOfPrint]

  • Mumps virus (MuV) encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleocapsid protein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, N-terminal domain (PN), oligomerization domain (PO) and C-terminal domain (PC). While PN is known to relax the NP-bound RNA genome, the roles of PO and PC are not clear. In this study, we investigated the roles of PO and PC in viral RNA synthesis using mutational analysis and a minigenome system. We have found that PN and PC functions can be trans-complemented. However, this complementation requires PO, indicating that PO is essential for P function. Using this trans-complement system, we have found that P forms parallel dimers (PN to PN and PC to PC). Furthermore, we have found that residues R231, K238, K253 and K260 in the Po were critical for P's functions. We have identified PC as the domain that interacts with L. These results provide structure-function insights into the role of MuV P.

    Significance MuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows domains of P to complement in trans. Using this system, we confirmed that MuV P forms parallel dimers. Understanding viral RNA synthesis will allow design of better vaccines and development of antivirals.

  • Novel reassortant human-like H3N2 and H3N1 influenza A viruses detected in pigs are virulent and antigenically distinct from endemic viruses [PublishAheadOfPrint]

  • Human-like swine H3 influenza A viruses (IAV) were detected by the USDA surveillance system. We characterized two novel swine human-like H3N2 and H3N1 viruses with HA genes similar to human seasonal H3 strains and the internal genes closely related to 2009 H1N1 pandemic viruses. The H3N2 NA was of the contemporary human N2 lineage, while the H3N1 NA was of the classical swine N1 lineage. Both viruses were antigenically distant from swine H3 viruses that circulate in the U.S. and from swine vaccine strains, and also showed antigenic drift from human seasonal H3N2. Their pathogenicity and transmission in pigs were compared to a human H3N2 with common HA ancestry. Both swine human-like H3 viruses efficiently infected pigs and transmitted to indirect contacts, whereas the human H3N2 was much less efficient. To evaluate the role of genes from the swine isolates on their pathogenesis, reverse genetics-generated reassortants between the swine human-like H3N1 and the seasonal human H3N2 were tested in pigs. Gene segment contribution to virulence was complex with the swine HA and internal genes showing effect in vivo. The experimental infections indicate that these novel H3 viruses are virulent and can sustain onward transmission in pigs, and the naturally occurring mutations in the HA were associated with antigenic divergence from H3 IAV from human and swine. Consequently, these viruses could have a significant impact on the swine industry if they cause more widespread outbreaks, and the potential risk of these emerging swine IAV to humans should be considered.

    IMPORTANCE Pigs are important hosts in the evolution of influenza A viruses (IAV). Human-to-swine transmissions of IAV have resulted in the circulation of reassortant viruses containing human-origin genes in pigs, greatly contributing to the diversity of IAV in swine worldwide. New human-like H3N2 and H3N1 viruses that contain a mix of human and swine gene segments were recently detected by the USDA surveillance system. The human-like viruses efficiently infected pigs and resulted in onward airborne transmission, likely due to multiple changes identified between human and swine H3 viruses. The human-like swine viruses are distinct from contemporary U.S. H3 swine viruses and from the strains used in swine vaccines, which could have a significant impact on the swine industry due to lack of population immunity. Additionally, public health experts should consider appropriate risk assessment for these emerging swine H3N1 for the human population.

  • Reorganization of nuclear pore complexes and lamina in late-stage parvovirus infection [PublishAheadOfPrint]

  • Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with decrease of lamin A/C on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity of NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina presumably to facilitate the late-stage infection.

  • Evolutionary dynamics and global diversity of influenza A virus [PublishAheadOfPrint]

  • The increasing number of zoonotic infections caused by influenza A virus (IAV) subtypes of avian origin in recent years (e.g. H5N1 and H7N9) underscores the need to better understand the factors driving IAV evolution and diversity. To evaluate the current feasibility of global analyses to contribute to this aim, we evaluated information in the public domain to explore IAV evolutionary dynamics including, nucleotide substitution rates and selection pressures, using 14 IAV subtypes in 32 different countries over a 12-year period (2000 nndash; 2011). Using geospatial information from 39,785 IAV strains, we examined associations between subtype diversity and socio-economic, biodiversity, and agricultural indices. Our analyses showed that nucleotide substitution rates for 11 of the 14 evaluated subtypes tended to be higher in Asian countries, particularly those in East Asia, compared to Canada and the United States. Similarly at a regional level, subtypes H5N1, H5N2, and H6N2 exhibited significantly higher substitution rates in East Asia compared to North America. In contrast, the selection pressures (measured as dN/dS ratios) acting on individual subtypes showed little geographic variation. We found that the strongest predictors for the detected subtype diversity at the country level were reporting effort and healthcare spending (an indicator of economic development). Our analyses also identified major global gaps in IAV reporting (including lack of sequences submitted from large portions of Africa and South America, and lack of geolocation information) and broad subtype testing which, until addressed, will continue to hinder efforts to track the evolution and diversity of IAV around the world.

    IMPORTANCE In recent years, an increasing number of influenza A virus (IAV) subtypes including H5N1, H7N9, and H10N8 have been detected in humans. Their high fatality rates have led to an increased urgency to better understand where and how novel pathogenic influenza strains emerge. Our findings showed that mutational rates of 11 commonly encountered subtypes were higher in East Asian countries compared to those in North America, suggesting there may be a greater risk for the emergence of novel pathogenic strains in East Asia. In assessing the potential drivers of IAV subtype diversity, our analyses confirmed that reporting effort and healthcare spending were the best predictors of observed subtype diversity at the country level. These findings underscore the need to increase sampling and reporting efforts for all subtypes in many under-sampled countries throughout the world.

  • Positive selection in CD8+ T-cell epitopes of influenza nucleoprotein revealed by a comparative analysis of human and swine viral lineages [PublishAheadOfPrint]

  • Numerous experimental studies have demonstrated that CD8+ T-cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T-cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses, and then compare the evolution of these epitopes in parallel lineages of human and swine influenza that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in the NP of human versus swine influenza, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions to human influenza NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza NP, sites in T-cell epitopes evolve more slowly than non-epitope sites, presumably because these epitopes are under higher inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T-cells in human influenza NP, and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint.

    IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse viral strains. CD8+ T-cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza evolution. Although many studies have shown that T-cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza evolves under positive selection to escape T-cells. Here we use comparisons of human and swine influenza to rigorously demonstrate that human influenza evolves under pressure to fix mutations in nucleoprotein that promote escape from T-cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the "trunk" of the phylogenetic tree. Overall, our results show that CD8+ T-cells targeting nucleoprotein play an important role in shaping influenza evolution.

  • Viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion. [PublishAheadOfPrint]

  • To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. Taken together this study provides a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.

    IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase (RdRp) in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. Here we identified a RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity, yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion, and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.

  • L-particles transmit viral proteins from HSV-1-infected mDCs to uninfected bystander cells inducing CD83 down-modulation [PublishAheadOfPrint]

  • Mature dendritic cells (mDCs) are known as the most potent antigen presenting cells (APCs) since they are also able to prime/induce naiiuml;ve T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have co-stimulatory properties, is targeted by herpes simplex virus type I (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in down-modulation of CD83 resulting in reduced T cell stimulation. In this study we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. Presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L-particles. These L-particles are generated during lytic replication in parallel to full virions, called H-particles. L-particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L-particles indeed reduced CD83 expression on uninfected bystander DCs providing for the first time evidence that functional viral proteins are transmitted via L-particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 down-modulation.

    IMPORTANCE HSV-1 has evolved a number of strategies to evade the host's immune system. Amongst others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L-particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses like human cytomegalovirus (HCMV), Epstein-Barr-Virus and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L-particles revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L-particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.

  • Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine [PublishAheadOfPrint]

  • Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however a recent report showed that equine influenza A viruses (IAV) can be isolated from pigs, although an established infection has not been observed so far in this host. To gain insight into the possibility of H3N8 avian IAV to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian and seal) were performed. As positive control, a H3N2 swine influenza virus A was used. While equine and canine viruses hardly replicated in the respiratory apparatus of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against HA could not be detected after infection by hemaglutination inhibition test (HAI) with the avian and seal virus. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAV from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using HAI as diagnostic tool.

    IMPORTANCE SECTION Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we could not detect specific antibodies against HA in any H3N8-infected pigs. Therefore, special attention should be focused towards viruses of the H3N8 subtype as they could behave as stealth viruses in pigs.

  • Host Gene Expression is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BART Region [PublishAheadOfPrint]

  • In Epstein-Barr virus infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs and the spliced and polyadenylated exons form nuclear non-protein coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells, however myc activation was confirmed through use of a myc responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full length cDNA clone of one of the BART isoforms was obtained and expressed in the EBV negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells where the BARTs are exclusively nuclear and do not encode for proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system.

    IMPORTANCE Infection with Epstein-Barr virus is nearly ubiquitous in the human population; however it does contribute to the formation of multiple types of cancer. In immune compromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV positive gastric carcinoma does not require immune suppression and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first data to show the highly expressed polyadenylated BART viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

  • Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies [PublishAheadOfPrint]

  • Cocktails of monoclonal antibodies (MAbs) that target the Ebola virus (EBOV) surface glycoprotein (GP) are effective in non-human primate models and have been used under emergency compassionate treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, as well as the related MB-003 cocktails, have yet to be identified. Other binding properties that define how each MAb is functionally interacting with GP nndash; such as affinity, epitope conservation, and epitope accessibility nndash; also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAb's specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds non-identical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus.

    IMPORTANCE Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected over ten times more people in Western Africa over the last year than all previous EVD outbreaks combined, with a number cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014 fifteen patients were treated with ZMapp or ZMAb under compassionate use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

  • A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription [PublishAheadOfPrint]

  • Influenza A virus (IAV) undergoes RNA transcription by a unique capped mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1 and PB2 proteins. But, how the viral RdRp utilizes cellular factors for viral transcription is not clear. Previously, we have conducted a genome-wide pooled shRNA screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a mini-replicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provides a target for new antivirals.

    IMPORTANCE Influenza virus is an important human pathogen which causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to high mutation rate of the virus, antiviral drugs targeting at viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the ribosomal RNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A viral transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for viral transcription and replication. Furthermore, we reported that RRP1B is crucial for RdRp binding to cellular capped mRNA, which is a critical step of virus transcription. Our study not only provides a deeper understanding of influenza virus-host interplay but also suggests a potential target for antiviral drug development.

  • In vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector [PublishAheadOfPrint]

  • Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. While blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for Pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum using a far-western blot method. Pull-down and immunofluorescence binding assays, and surface plasmon resonance were used to confirm and characterize CP nndash; APN interaction. PEMV virions and a peptide comprised of PEMV coat protein fused to GFP (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles (BBMV), binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut, and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W.O. Sparks, W.A. Miller, B.C. Bonning, Virology 401:107-16, 2010, doi: 10.1016/j.virol.2010.02.009) support identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV nndash; APN interaction designed to block plant virus transmission and to suppress aphid populations.

    IMPORTANCE A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers rely heavily on the application of chemical insecticides to manage both aphids and aphid-vectored plant viral disease. To increase our understanding of plant virus-aphid vector interaction, we provide in vitro evidence supporting earlier in vivo work for identification of a receptor protein in the aphid gut called aminopeptidase N, which is responsible for entry of the plant virus Pea enation mosaic virus into the pea aphid vector. Enrichment of proteins found on the surface of the aphid gut epithelium resulted in identification of this first aphid gut receptor for a plant virus. This discovery is particularly important as disruption of plant virus binding to such a receptor may enable development of a non-chemical strategy for controlling aphid-vectored plant viruses to maximize food production.

  • GENOME-WIDE SCREEN REVEALS VCP REQUIREMENT FOR CORONAVIRUS EXIT FROM ENDOSOMES. [PublishAheadOfPrint]

  • Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by SARS and the emerging MERS. Cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. 30 of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics.

    IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics.

  • Sinorhizobium meliloti phage {Phi}M9 defines a new group of T4-superfamily phages with unusual genomic features, but a common T=16 capsid [PublishAheadOfPrint]

  • Relatively little is known about the phages that infect agriculturally-important, nitrogen-fixing rhizobial bacteria. Here, we report the genome and cryo-EM structure of the Sinorhizobium meliloti-infecting T4 superfamily phage M9. This phage, along with its close relative Rhizobium phage vB_RleM_P10VF, defines a new group of T4 superfamily phages. These phages are distinctly different from the recently-characterized cyanophage-like S. meliloti phages of the M12 group. Structurally, M9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the M9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. M9 also shares the T=16 capsid architecture with the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, M9 and S. meliloti phage M12 share a small number of ORFs that appear to encode structural proteins involved in interaction with the host, and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins and glycanases that cleave host exopolysaccharide.

    IMPORTANCE Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic, nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil-bacteria/phage dynamics are genetic, molecular and structural characterization of these groups of phages. The T4-superfamily phages are among the most complex phages: they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study of the Sinorhizobium meliloti-infecting T4 superfamily phage M9 provides new insight into the diversity of this family. The comparison of structure-related genes in both M9 and the Sinorhizobium meliloti-infecting T4-superfamily phage M12, which comes from a completely different lineage of these phages allows the identification of host-infection related factors.

  • Middle East respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting nuclear-transcribed mRNAs but spares mRNAs of cytoplasmic origin [PublishAheadOfPrint]

  • The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a common property to inhibit host gene expression at the post-transcriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 employed a strikingly different strategy that selectively targeted translationally-competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation, but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point towards a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1.

    Importance Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of MERS-CoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. The nonstructural protein 1 (nsp1) of CoVs is one such protein that inhibits host gene expression and is a major virulence factor. This study presents evidence for a strategy employed by MERS-CoV nsp1 to inhibit host gene expression that has not been described previously for any viral protein. The present study represents a meaningful step towards a better understanding of the factors and molecular mechanisms governing the virulence and pathogenesis of MERS-CoV.

  • Out of the reservoir: Phenotypic and genotypic characterization of a novel cowpox virus isolated from a common vole [PublishAheadOfPrint]

  • The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV-reservoir. We characterized a CPXV isolated during a large scale screening from a feral common vole. A comparison of the full-length DNA-sequence of this CPXV-strain with a highly virulent pet rat CPXV-isolate showed a sequence identity of 96%, including a large additional ORF of about 6,000 nucleotides, which is absent in the reference CPXV-strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion bodies (ATI) with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV-strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV-strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species.

    IMPORTANCE We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes only present in a reservoir isolate can now be further analyzed. These studies allow, therefore, unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission, and the zoonotic threat that these viruses represent.

  • Differences in processing determinants of non-structural polyprotein and in the sequence of non-structural protein 3 affect neurovirulence of Semliki Forest virus. [PublishAheadOfPrint]

  • The A7 (74) strain of Semliki Forest virus (SFV, genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7 (74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3 encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions of amino acid residues 534 (P4-position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534 to Arg substitution specifically activated this cleavage, while a Val1052 to Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV.

    IMPORTANCE SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes was confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.

  • Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane domain (7TMD) proteins, nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells and in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20 encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMDs protein, which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells.

    IMPORTANCE Human Cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells that represent a key target for viral dissemination and replication in the host, and contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a post-entry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis, and help lead to the design of new antiviral strategies able to exploit these functions.

  • Selective Removal Of FG-Repeat Domains From The Nuclear Pore Complex By Enterovirus 2Apro [PublishAheadOfPrint]

  • Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport the mechanisms and consequences of Nup98 proteolysis have been examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2Apro). Incubation of bacterially expressed or in vitro translated Nup98 with 2Apro results in proteolytic cleavage at multiple sites in vitro indicating that 2Apro cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2Apro proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N-terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C-terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG-repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains overall NPC structure and leaves other transport pathways unaffected.

    IMPORTANCE Enteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N-terminus of Nup98 from the NPC, while the C-terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C-terminus of Nup98 remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their composition.

  • The role of the DNA sensor STING in protection from lethal infection following corneal and intracerebral challenge with HSV-1. [PublishAheadOfPrint]

  • STING is a protein in the cytosolic DNA and cyclic dinucleotide sensor pathway that is critical for the initiation of innate responses to infection by various pathogens. Consistent with this, HSV-1 causes invariable and rapid lethality in STING-deficient (STING-/-) mice following intravenous (iv) infection. In this study, using real-time bioluminescence imaging and virological assays, as expected, we demonstrated that STING-/- mice support greater replication and spread in ocular tissues and the nervous system. In contrast, they did not succumb to challenge via the corneal route even with high titers of a virus that was routinely lethal to STING-/- mice by the iv route. Corneally-infected STING-/- mice also showed increased periocular disease, increased corneal and trigeminal ganglia titers although no difference in brain titers. They also showed elevated expression of TNFaalpha; and CXCL9 relative to control mice, but surprisingly modest changes in type I interferon expression. Finally, we also showed that HSV strains lacking the ability to counter autophagy and the PKR-driven antiviral state had near-wild-type virulence following intracerebral infection of STING-/- mice. Together, these data show that while STING is an important component of host resistance to HSV in the cornea, its previously-shown immutable role in mediating host survival by the iv route was not recapitulated following a mucosal infection route. Furthermore, our data are consistent with the idea that HSV counters STING-mediated induction of the antiviral state and autophagy response, both of which are critical factors for survival following direct infection of the nervous system.

    IMPORTANCE HSV infections represent an incurable source of morbidity and mortality in humans, and are especially severe in neonatal and immune-compromised populations. A key step in development of an immune response is the recognition of microbial components within infected cells. The host protein STING is important in this regard for the recognition of HSV DNA, and the subsequent triggering of innate responses. STING was previously shown to be essential for protection against lethal challenge from intravenous HSV-1 infection. In this study we show that the requirement for STING depends on the infection route. In addition, STING is important for appropriate regulation of the inflammatory response in the cornea, and our data are consistent with the idea that HSV modulates STING activity through inhibition of autophagy. Our results elucidate the importance of STING in host protection from HSV-1, and demonstrate the redundancy of host protective mechanisms, especially following mucosal infection.

  • The infectious bursal disease virus VP3 upregulates VP1-mediated RNA-dependent RNA replication [PublishAheadOfPrint]

  • Genome replication is a critical step in virus life cycles. Here, we have analyzed the role of the infectious bursal disease virus (IBDV) VP3, a major component of IBDV ribonucleoprotein complexes, on the regulation of VP1, the virus-encoded RNA-dependent RNA polymerase (RdRP). Data show that VP3, as well as a peptide mimicking its C-terminal domain, efficiently stimulate the ability of VP1 to replicate synthetic single stranded RNA templates containing the 3rrsquo; -UTRs from the IBDV genome segments.

  • Herpesvirus saimiri microRNAs Preferentially Target Host Cell-Cycle Regulators [PublishAheadOfPrint]

  • In latently-infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover functions of miR-HSURs, we identified mRNA targets in infected cells using High-Throughput Sequencing of RNA Isolated by Crosslinking Immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs in the host genome, but few in the HVS genome. Gene Ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional co-activator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER) localized chaperone facilitating maturation of the Major Histocompatibility Complex I (MHC I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell-cycle progression, leading to reduced phosphorylation of its substrate cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency.

    IMPORTANCE Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic nndash;herpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge about virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell-cycle regulators, as well as antiviral response factors. Our work provides further insight into function of herpesviral miRNAs in virally-induced oncogenesis and latency.

  • Functional comparison of Mx1 from two different mouse species reveals the involvement of loop L4 in the antiviral activity against influenza A viruses [PublishAheadOfPrint]

  • The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mus musculus Mx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza activity of Mx1 from Mus musculus A2G with its ortholog from Mus spretus. We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Sequence comparison of M. musculus with M. spretus Mx1 revealed 25 amino acid differences, over half of which are present in the GTPase domain and two in loop L4. However, the in vitro GTPase activity of Mx1 from the two mouse species was similar. Replacing one of the residues in loop L4 in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented binding of Mx1 to influenza A virus nucleoprotein and hence abolished antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses.

    IMPORTANCE Mx proteins are evolutionary conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of Mx genes from two species, that diverged relatively recently in evolution, can provide novel insight into these mechanisms. We show that both Mus musculus A2G Mx1 and Mus spretus Mx1 target influenza nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was reported recently for primate MxA. This indicates that human and mouse Mx proteins, which diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common mechanism.

  • Transition towards Human Cytomegalovirus Susceptibility in Early Human Embryonic Stem Cells - Derived Neural Precursors [PublishAheadOfPrint]

  • Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem-cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, towards HCMV-susceptibility in early hESC-derived neural precursors. We further revealed the role of PDGFRaalpha; as a determinant of the developmentally-acquired HCMV susceptibility.

  • Breast milk of HIV-positive mothers has potent and species-specific in vivo HIV inhibitory activity [PublishAheadOfPrint]

  • Despite the nutritional and health benefits of breast milk, breast milk can serve as a vector for mother-to-child HIV transmission. Most HIV-infected infants acquire HIV through breastfeeding. Paradoxically, most infants breastfed by HIV-positive women do not become infected. This is potentially attributed to anti-HIV factors in breast milk. Breast milk of HIV-negative women can inhibit HIV infection. However, the HIV inhibitory activity of breast milk from HIV-positive mothers has not been evaluated. In addition, while significant differences in breast milk composition between transmitting and non-transmitting HIV-positive mothers have been correlated with transmission risk, the HIV inhibitory activity of their breast milk has not been compared. This knowledge may significantly impact the design of prevention approaches in resource-limited settings that do not deny infants of HIV-positive women the health benefits of breast milk. Here, we utilized BLT humanized mice to evaluate the in vivo HIV inhibitory activity of breast milk obtained from HIV-positive transmitting and non-transmitting mothers. We also assessed the species-specificity and biochemical characteristics of milk's in vivo HIV inhibitory activity and its ability to inhibit other modes of HIV infection. Our results demonstrate that breast milk of HIV-positive mothers has potent HIV inhibitory activity and indicate that breast milk can prevent multiple routes of infection. Most importantly, this activity is unique to human milk. Our results also suggest multiple factors in breast milk may contribute to its HIV inhibitory activity. Collectively, our results support current recommendations that HIV-positive mothers in resource-limited settings exclusively breastfeed in combination with antiretroviral therapy.

    IMPORTANCE Approximately 240,000 children become infected with HIV annually, the majority via breastfeeding. Despite daily exposure to virus in breast milk, most infants breastfed by HIV-positive women do not acquire HIV. The low risk of breastfeeding-associated HIV transmission is likely due antiviral factors in breast milk. It is well-documented that breast milk of HIV-negative women can inhibit HIV infection. Here, we demonstrate, for the first time, that breast milk of HIV-positive mothers (non-transmitters and transmitters) inhibits HIV transmission. We also demonstrate that breast milk can prevent multiple routes of HIV acquisition and that this activity is unique to human milk. Collectively, our results support current guidelines which recommend that HIV-positive women in resource-limited settings exclusively breastfeed in combination with infant or maternal antiretroviral therapy.

  • Site-specific recombination by SSV2 integrase: substrate requirement and domain functions [PublishAheadOfPrint]

  • SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (IntSSV2). IntSSV2 alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1-80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165-328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81-164). It is worth noting that an N-terminally truncated mutant of IntSSV2 (residues 81-328), which corresponded to the putative product of the 3rrsquo; -end sequence of the IntSSV2 gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length IntSSV2 except for the slightly reduced recombination activity. Our results suggest that, unlike integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion.

    IMPORTANCE Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV-type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision, and reveal how different parts of the target DNA and the enzyme serve their roles in the processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.

  • Type I interferons triggered through the TLR3-TRIF pathway control Coxsackievirus A16 infection in young mice [PublishAheadOfPrint]

  • Coxsackievirus A16 (CVA16) is one of the major etiological agents that cause hand, foot and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with EV71 infection, here we show that IFN- or iNKT cell deficiency did not affect the disease development and survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in mice developing more severe disease, and they had a lower survival rate than wild-type mice. Similarly, deficiency of TLR3 and TRIF, but not other pattern recognition receptors, led to decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-bbeta; treatment was also found to compensate for TRIF deficiency in mice and decreased disease severity and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection.

    IMPORTANCE Hand, foot and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71 but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet be elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.

  • Equine herpesvirus type 1 (EHV-1) enhances viral replication in CD172a+ monocytic cells upon adhesion to endothelial cells [PublishAheadOfPrint]

  • Equine herpesvirus type 1 (EHV-1) is a main cause of respiratory disease, abortion and encephalomyelopathy in horses. Monocytic cells (CD172a+) are the main carrier cells of EHV-1 during primary infection and are proposed to serve as a llsquo;Trojan horse' to facilitate the dissemination of EHV-1 to target organs. However, the mechanism by which EHV-1 is transferred from CD172a+ cells to endothelial cells (EC) remains unclear. The aim of this study was to investigate EHV-1 transmission between these two cell types. We hypothesized that EHV-1 employs specific strategies to promote the adhesion of infected CD172a+ cells to EC to facilitate EHV-1 spread. Here we demonstrated that EHV-1 infection of CD172a+ cells resulted in a 3 to 5-fold increase in adhesion to EC. Antibody-blocking experiments indicated that aalpha;4bbeta;1, aalpha;Lbbeta;2 and aalpha;Vbbeta;3 integrins mediated adhesion of infected CD172a+ cells to EC. We showed that integrin-mediated PI (3)K and ERK/MAPK signaling pathways were involved in EHV-1-induced CD172a+ cell adhesion at early time of infection. EHV-1 replication was enhanced in adherent CD172a+ cells, which correlates with the production of TNF-aalpha;. In the presence of neutralizing antibodies, approximately 20% of infected CD172a+ cells transferred cytoplasmic material to uninfected EC and 0.01% of infected CD172a+ cells transmitted infectious virus to neighbouring cells. Our results demonstrated that EHV-1 infection induces adhesion of CD172a+ cells to EC, which enhances viral replication, but that transfer of viral material from CD172a+ cells to EC is a very specific and rare event. These findings give new insights in the complex pathogenesis of EHV1.

    Author summary Equine herpesvirus type 1 (EHV-1) is a highly prevalent pathogen worldwide, causing frequent outbreaks of abortion and myeloencephalopathy, even in vaccinated horses. After primary replication in the respiratory tract, EHV-1 disseminates via cell-associated viremia in peripheral blood mononuclear cells (PBMC) and subsequently infects the endothelial cells (EC) of the pregnant uterus or central nervous system, leading in some cases to abortion and/or neurological disorders. Recently, we demonstrated that CD172a+ monocytic carrier cells serve as a llsquo;Trojan horse' to facilitate EHV-1 spread from blood to target organs. Here, we investigated the mechanism underlying the transmission of EHV-1 from CD172a+ cells to EC. We demonstrated that EHV-1 infection induces cellular changes in CD172a+ cells promoting their adhesion to EC. We found that both cell-to-cell contacts and the secretion of soluble factors by EC activate EHV-1 replication in CD172a+ cells. This facilitates transfer of cytoplasmic viral material to EC, resulting mainly in a non-productive infection. Our findings give new insights on how EHV-1 may spread to EC of target organs in vaccinated horses.

  • Identification And Mechanism Of Action Of A Novel Small Molecule Inhibitor Of Arenavirus Multiplication [PublishAheadOfPrint]

  • Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is an important neglected human pathogen. There are no licensed arenavirus vaccines and current anti-arenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel anti-arenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high throughput screen (HTS) to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism of action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein (vRNP) into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the trans-membrane domain of GP2 as critical for virus susceptibility to F3406.

    IMPORTANCE Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in their endemic areas. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of FDA-licensed arenavirus vaccines and current anti-arenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.

  • Unique determinants of neuraminidase inhibitor resistance among N3, N7, and N9 avian influenza viruses [PublishAheadOfPrint]

  • Human infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI-resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7 and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with 7 internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In fluorescence-based NA inhibition assay we identified 3 categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir); i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering); ii) subtype-independent substitutions (E119G/V and/or D and R292K); and iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically.

    IMPORTANCE The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). As most studies have focused on NAI-resistance in human influenza viruses, here we investigated the molecular changes in NA that could confer NAI-resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7 and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype-specific. The findings indicate that some resistance markers are common across NA subtypes but other markers needs to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza infection and an essential component of pandemic preparedness.

  • Vaccine-induced SIV-specific CD8+ T-cell responses focused on a single Nef epitope select for escape variants shortly after infection [PublishAheadOfPrint]

  • Certain MHC class I (MHC-I) alleles (e.g. HLA-B*27) are enriched among HIV-1-infected individuals who suppress viremia without treatment (termed "elite controllers"; ECs). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control SIV replication. Given similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08+ animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08+ macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlates analysis revealed that CD8+ T-cells targeting Nef RL10 correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced NefRL10-specific CD8+ T-cell response would facilitate the development of elite control in Mamu-B*08+ animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08+ animals with nef inserts in which Nef RL10 was either left intact (Group 1) or disrupted by mutations (Group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in Group 1 developed Nef RL10-specific CD8+ T-cells. These vaccine-induced effector memory CD8+ T-cells did not prevent infection. Escape variants emerged rapidly in the Group 1 vaccinees and, ultimately, the number of ECs was similar in Groups 1 and 2. High frequency vaccine-induced CD8+ T-cells focused on a single conserved epitope, therefore, did not prevent infection or increase the incidence of elite control in Mamu-B*08+ macaques.

    IMPORTANCE Since elite control of chronic phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights as to how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8+ T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08+ rhesus macaquesnndash;a model of HLA-B*27-mediated elite control. Here we investigated if a monotypic vaccine-induced CD8+ T-cell response targeting the conserved, "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08+ monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8+ T-cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8+ T-cell responses against more than one epitope. Together, these results underscore the complexity and multi-dimensional nature of virologic control of lentivirus infection.

  • Intracellular signaling and desmoglein 2 shedding triggered by human adenoviruses Ad3, Ad14, and Ad14P1 [PublishAheadOfPrint]

  • We recently discovered that DSG2 is a receptor for human adenovirus species B serotypes Ad3, 7, 11, and 14. Ad3 is considered to be a widely distributed human pathogen. Ad3 binding to DSG2 triggers the transient opening of epithelial junctions. Here we further delineate the mechanism that leads to DSG2 mediated epithelial junction opening in cells exposed to Ad3 and recombinant Ad3 fiber proteins. We identified an Ad3 fiber knob-dependent pathway that involves the phosphorylation of MAP kinases triggering the activation of the matrix-metalloproteinase ADAM17. ADAM17 in turn cleaves the extracellular domain of DSG2 that links epithelial cells together. The shed DSG2 domain can be detected in cell culture supernatant and also in serum of mice with established human xenograft tumors. We then extended our studies to Ad14 and Ad14P1. Ad14 is an important research and clinical object, because of the recent appearance of a new, more pathogenic strain (Ad14P1). In a human epithelial cancer xenograft model, Ad14P1 showed more efficient viral spread and oncolysis than Ad14. Here we tested the hypothesis whether a mutation in the Ad14P1 fiber knob could account for the differences between the two strains. While our X-ray crystallography studies suggested an altered 3D structure of the Ad14P1 fiber knob in the F-G loop region, this did not significantly change the fiber knob affinity to DSG2 nor the intracellular signaling and DSG2 shedding in epithelial cancer cells.

    IMPORTANCE A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 not only allows the virus to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber knob domain and triggers intracellular signaling that culminates in the cleavage of the extracellular domain of DSG2 thereby disrupting DSG2 homodimers between epithelial cells. We confirmed this pathway with a second DSG2-interacting serotype, Ad14 and its recently emerged stain Ad14P1. These new insights in basic adenovirus biology can be employed to develop novel drugs to treat adenovirus infection as well as be used as tools for gene delivery into epithelial tissues or epithelial tumors.

  • AAV2 Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome [PublishAheadOfPrint]

  • Adeno-associated virus type-2 is known to inhibit replication of herpes simplex virus type-1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep-helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/78 to the HSV-1 genome.

  • The Dynamics of HIV-1 RNA Near the Plasma Membrane During Virus Assembly [PublishAheadOfPrint]

  • To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognized stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane and followed the population of the photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently for a few minutes. The presence of Gag significantly increased the time RNAs stayed near the plasma membrane; most of the RNAs were detected after 30 minutes. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that was packaged into assembling viral complexes. By tagging Gag with a blue fluorescent protein, we observed that only a portion, ~13-34%, of the HIV-1 RNAs that reached the membrane was recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with Gag expression level. Our studies reveal HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions.

    IMPORTANCE Nascent HIV-1 particles assemble on the plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we have labeled and followed the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stay near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNA stayed at the plasma membrane for more than 15-60 minutes in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately one tenth to one third of the RNAs that reached plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained the temporal information of RNA-Gag interactions that lead to RNA encapsidation.

  • A Conserved Gammaherpesvirus Cyclin Specifically Bypasses Host p18INK4c To Promote Reactivation From Latency [PublishAheadOfPrint]

  • Gammaherpesviruses encode homologs of cellular genes, including a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors, p27Kip1 and p18INK4c, two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, the viral cyclin was dispensable for reactivation in mice lacking p27Kip1 and p18INK4c. Further analysis revealed that genetic ablation of only p18INK4c alleviated the requirement for the viral cyclin reactivation from latency. p18INK4c regulated reactivation in a dose-dependent manner, such that the viral cyclin was dispensable in p18INK4c heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18INK4c function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data identify that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin dependent kinase inhibitor p18INK4c, revealing an unanticipated specificity between a HV cyclin and a single cyclin-dependent kinase inhibitor.

    IMPORTANCE The gammaherpesviruses (HVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many HVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the HVs resist this inhibition. We used a mouse HV that either contains or lacks a viral cyclin, to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies identified that the viral cyclin was required for optimal function in normal mice, but that the viral cyclin was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor, and point to new methods to intervene with viral cyclins.

  • Improvement of HIV-1 and HTLV-1 Replication-Dependent Vectors via Optimization of Reporter Gene Reconstitution and Modification with Intronic shRNA [PublishAheadOfPrint]

  • Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T-lymphtropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in co-cultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents reporter gene expression in the transfected cells, but permits its expression in target cells after infection. However, the gamma-globin intron is not removed efficiently by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via miR30-based shRNAs. Using two optimized fluorescence-reporter vectors and flow cytometry, we have determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase is dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify infection with higher sensitivity in lymphocytes and in primary cells, and allow the detection and quantitation of multiply-infected cells, providing better tools to study retroviral cell-mediated infection.

    Importance The human pathogenic retroviruses, HTLV-1 and HIV-1, can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current report, we have generated vectors that are significantly improved from the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in mixed culture cells. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one step transfection/infection assay.

  • Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity [PublishAheadOfPrint]

  • The cytoplasmic tails of human and simian immunodeficiency virus envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface.

    Importance This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals.

  • MicroRNA-attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma [PublishAheadOfPrint]

  • Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem severely restricting viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4 that causes lethal encephalitis in mice is able to infect and replicate independent of IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124, a derivative of SFV4 with restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA-124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region, and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated SFV A7 (74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 in C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain, and was amplified in the tumor resulting in significant tumor growth inhibition and improved survival.

    IMPORTANCE Although progress has been made in development of replicative oncolytic viruses, the overall therapeutic potency in clinical setting is still lacking. This could be at least partially dependent on IFN-I sensitivity of the viruses used. Here we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I-tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of glioma that is refractory to naturally attenuated therapy vector strains that are sensitive to IFN-I. This is the first evidence of orthotopic syngeneic mouse glioma eradication following peripheral alphavirus administration. Our findings indicate a clear benefit in harnessing the wild-type virus replicative potency in development of next generation oncolytic alphaviruses.

  • Actin-Modulating Protein Cofilin Is Involved in the Formation of Measles Virus Ribonucleoprotein Complex at the Perinuclear Region [PublishAheadOfPrint]

  • In measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and nucleocapsid (N), phospho (P)- and large proteins, assembles at the perinuclear region, and synthesizes viral RNAs. Cellular proteins involved in the formation of the RNP complex are largely unknown. In this study, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of the RNP complex formation results in the reduction of viral RNA synthesis. Cofilin phosphorylated on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing the RNP complex formation and viral RNA synthesis.

    IMPORTANCE Many RNA viruses induce within infected cells the structure called the ribonucleoprotein (RNP) complex in which viral RNA synthesis occurs. It is comprised of the viral genome and proteins including the viral RNA polymerase. Cellular proteins involved in the formation of the RNP complex are largely unknown. In this study, we show that cofilin, an actin-modulating host protein, binds to the measles virus (MV) nucleocapsid protein and plays an important role in the formation of the MV RNP complex and MV RNA synthesis. The phosphorylated form of cofilin, enzymatically inactive, is increased after MV infection, and preferentially associated with the RNP complex. Our finding with cofilin will help us better understand the mechanism by which the RNP complex is formed in virus-infected cells and develop new anti-viral drugs targeting the RNP complex.

  • Viral source-independent high susceptibility of dendritic cells to HTLV-1 infection compared to T lymphocytes. [PublishAheadOfPrint]

  • HTLV-1-infected CD4+ T-cells and dendritic cells (DCs) are present in peripheral blood from HTLV-1 carriers. While T-cell infection requires cell-cell contact, DCs might be infected with cell-free virus, at least in vitro. However, a thorough comparison of both cell types susceptibility to HTLV-1 infection using cell-associated and cell-free viral sources has not been performed. We first determined that human primary monocyte-derived dendritic cells (MDDCs) were more susceptible to HTLV-1 infection than their autologous lymphocyte counterparts after contact with chronically infected cells. Next, a comparison of infection efficiency using non-concentrated or concentrated supernatant from infected cells as well as purified viral biofilm was performed. Integrated provirus was found after exposition of MDDCs or primary lymphocytes to viral biofilm but not to viral supernatant. Using a large series of primary cell samples (n=21), we demonstrated a higher proviral load in MDDCs exposed to viral biofilm than in lymphocytes. This higher susceptibility is correlated to a higher expression of Neuropilin-1 on MDDCs than on autologous activated T lymphocytes. Moreover, we show that MDDCs infected with viral biofilm can transmit the virus to lymphocytes. In conclusion, MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro, supporting a model where DC infection might represent an important step during primo-infection in vivo.

    Importance HTLV-1 is able to infect several cell types but viral DNA is mainly found in T lymphocytes in vivo. This supports a model in which T lymphocytes are the main target of infection. However, during the primo infection of new individuals, incoming viruses might first encounter dendritic cells (DCs), the specialized immune cells responsible for the antiviral response of the host. HTLV-1 cell-free purified viruses can infect dendritic cells in vitro, while T-cells infection is restricted to cell-to-cell transmission. In order to understand the sequence of HTLV-1 dissemination, we undertook a direct comparison of both cell types susceptibility using cell-associated and cell-free viral sources. We report here that MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro and able to efficiently transmit the virus to lymphocytes. Our results suggest that DCs may represent a true viral reservoir, as being the first cell to be infected in vivo.

  • Human Papillomavirus Type 18 Genetic Variation and Cervical Cancer Risk Worldwide [PublishAheadOfPrint]

  • Human papillomavirus (HPV) type 18 is the second most carcinogenic HPV type after HPV16 and accounts for approximately 12% of squamous cell carcinoma (SCC) as well as 37% of adenocarcinoma (ADC) of the cervix worldwide. We aimed to evaluate the worldwide diversity and carcinogenicity of HPV18 genetic variants by sequencing the entire long control region (LCR) and the E6 open reading frame of 711 HPV18-positive cervical samples from 39 countries, taking advantage of the International Agency for Research on Cancer biobank. A total of 209 unique HPV18 sequence variants were identified that formed three phylogenetic lineages (A, B, and C). A and B lineages each divided into four sublineages, including a newly identified candidate B4 sublineage. The distribution of lineages varied by geographical region, with B and C lineages found principally in Africa. HPV18 (sub)lineages were compared between 453 cancer cases and 236 controls, as well as between 81 ADC and 160 matched SCC cases. In region-stratified analyses, there were no significant differences in the distribution of HPV18 variant lineages between cervical cancer cases and controls, or between ADC and SCC. In conclusion, our findings do not support the role of HPV18 (sub)lineages for discriminating cancer risk or explaining why HPV18 is more strongly linked with ADC than SCC.

    Importance: This is the largest and most geographically/ethnically diverse study of the genetic variation of HPV18 to date, providing a comprehensive reference for phylogenetic classification of HPV18 sublineages for epidemiological and biological studies.

  • Nef is dispensable for resistance of SIV-infected macrophages to CD8+ T cell killing. [PublishAheadOfPrint]

  • SIVnndash;specific CD8+ T cells kill SIVnndash;infected CD4+ T cells in an MHC Class I (MHCnndash;I) dependent manner. However, they are reportedly less efficient at killing SIVnndash;infected macrophages. Since the viral accessory protein, Nef, has been shown to downnndash;regulate MHCnndash;I molecules and enhance CTL evasion in HIVnndash;1 infected CD4+ T cells, we examined whether Nef played a role in protecting SIVnndash;infected macrophages from killing by SIVnndash;specific CD8+ T cells. To explore the role of Nef in CD8+ T cell evasion, we compared the ability of freshly sorted SIVnndash;specific CD8+ T cells to readily suppress viral replication or eliminate CD4+ T cells or monocytenndash;derived macrophages infected with SIV variants containing wildnndash;type (WT) or mutated nef genes. As expected, SIVnndash;specific CD8+ T cells suppressed viral replication and eliminated the majority of SIVnndash;infected CD4+ T cells and this killing was enhanced in CD4+ T cells infected with the nef variants. However, macrophages infected with nef variants that disrupt MHCnndash;I downnndash;regulation did not promote rapid killing by freshly isolated CD8+ T cells. These results suggest that mechanisms other than Nefnndash;mediated MHCnndash;I downnndash;regulation govern the resistance of SIVnndash;infected macrophages to CD8+ T cellnndash;mediated killing. This study has implications for viral persistence and suggests that macrophages may afford primate lentiviruses some degree of protection from immune surveillance.

    IMPORTANCE Myeloid cells are permissive for HIV/SIV replication in vitro and may contribute to viral persistence in vivo. While many studies have been geared to understanding how CD8+ T cells control viral replication in CD4+ T cells, the role of these cells in controlling viral replication in macrophages is less clear. Primary, unstimulated CD8+ T cells insignificantly suppress viral replication or eliminate SIVnndash;infected macrophages. Since the viral Nef protein downnndash;regulates MHCnndash;I and provides infected cells some degree of protection from CD8+ T cellnndash;mediated effector functions, we evaluated whether Nef may be contributing to the resistance of macrophages to CD8+ T cell suppression. Our results suggest that Nef is not involved in protecting infected macrophages from CD8+ T cell killing and suggest that other mechanisms are involved in macrophage evasion from CD8 surveillance.

  • REPLICATION-COMPETENT CONTROLLED HERPES SIMPLEX VIRUS [PublishAheadOfPrint]

  • We present the development and characterization of a replication-competent controlled Herpes Simplex Virus 1 (HSV-1). Replication-essential genes ICP4 and ICP8 of HSV-1 wild type strain 17syn+ were brought under the control of a dual-responsive gene switch. The gene switch comprises (1) a transactivator that is activated by a narrow class of antiprogestins including mifepristone and ulipristal and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (2) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of an administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells were only exposed to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of regulated gene ICP4 and late gene gC, which was not regulated. Similar findings were made in experiments with the mouse footpad infection model.

    IMPORTANCE The alpha-herpesviruses have long been considered as vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a not-attenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without a sacrifice in safety.

  • A molecular staple: D-loops in the I-domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly [PublishAheadOfPrint]

  • Bacteriophage P22, a dsDNA virus, has a non-conserved 124 amino acid residue accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This llsquo;I-domain' is involved in virus capsid size determination and stability, as well as protein folding. The NMR solution structure of the I-domain revealed the presence of a llsquo;D-loop', which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking we show the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as llsquo;molecular staples' at the icosahedral two-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging.

    Importance Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by inter-capsomer crosslinking, but most virus capsids are stabilized by alternate means. Here we show the I-domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I-domain allows for stabilizing interactions across the capsid two-fold axis of symmetry via a llsquo;D-loop'. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products including tubes are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.

  • Propranolol decreases proliferation of endothelial cells transformed by Kaposi sarcoma Herpesvirus and induces lytic viral gene expression [PublishAheadOfPrint]

  • Kaposi sarcoma (KS) is common in Africa, but economic constraints hinder successful treatment in most patients. Propranolol, a generic bbeta;-adrenergic antagonist, decreased proliferation of KS Herpesvirus- (KSHV-) infected cells. Down-regulation of cyclin A2 and cyclin-dependent kinase 1 (CDK1) recapitulated this phenotype. Additionally, propranolol induced lytic gene expression in association with down-regulation of CDK6. Thus, propranolol has diverse effects on KSHV-infected cells, and this generic drug has potential as a therapeutic agent for KS.

  • The breadth of expandable memory CD8+ T cells inversely correlates with residual viral loads in HIV elite controllers [PublishAheadOfPrint]

  • Previous studies have shown that elite controllers with minimal effector T cell responses harbor a low frequency, readily expandable, highly functional and broadly directed memory population. Here, we interrogated the in vivo relevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8+ T cells were expanded using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses compared to untreated progressors (p=0.01) as well as treated progressors (p=0.0003). Nef and Env-specific memory cells expanded poorly for all groups and their expanded breadths were indistinguishable between the groups (Nef, Kruskal- Wallis, p=0.9: Env, Kruskal-Wallis, p=0.6). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses inversely correlated with residual viral load (r=-0.6, p=0.009). Together these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8+ T cell feature that would be desirable in a vaccine induced T cell response.

    IMPORTANCE Many studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. Identifying key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes known as central memory CD8+ T lymphocyte is at least partially involved in the durable control HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8+ T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine induced CD8+ T cell responses.

  • Enhanced influenza VLP vaccination with a structurally optimized RIG-I agonist as adjuvant [PublishAheadOfPrint]

  • The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5rrsquo; pppRNA RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) expressing influenza H5N1 hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with influenza H5N1.. M8-VLP immunization also led to long term protective responses against influenza infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza replication in lungs when compared with approved or experimental adjuvants Alum, AddaVax, and poly(I:C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza challenge.

    IMPORTANCE The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5rrsquo; pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza antibody titers and protected animals from the lethal influenza challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.

  • Induction of Heme Oxygenase-1 Deficiency and Associated Glutamate-Mediated Neurotoxicity is a Highly Conserved HIV Phenotype of Chronic Macrophage Infection that is Resistant to Antiretroviral Therapy [PublishAheadOfPrint]

  • Expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly reduced in brain prefrontal cortex of HIV-positive individuals with HIV-associated neurocognitive disorders (HAND). Furthermore, this HO-1 deficiency correlates with brain viral load, markers of macrophage activation, and type I interferon responses. In vitro, HIV replication in monocyte-derived macrophages (MDM) selectively reduces HO-1 protein and RNA expression and induces production of neurotoxic levels of glutamate; correction of this HO-1 deficiency reduces neurotoxic glutamate production without an effect on HIV replication. We now demonstrate that macrophage HO-1 deficiency and associated neurotoxin production is a conserved feature of infection with macrophage-tropic HIV-1 strains that correlates closely with the extent of replication, and extends to HIV-2 infection. We further demonstrate that this HO-1 deficiency does not depend specifically upon the HIV-1 accessory genes nef, vpr or vpu, but rather on HIV replication, even when markedly limited. Finally, antiretroviral therapy (ART) applied to MDM after HIV infection is established does not prevent HO-1 loss and associated neurotoxin production. This work defines a predictable relationship between HIV replication, HO-1 loss, and neurotoxin production in MDM that likely reflects processes in place in the HIV-infected brain of individuals receiving ART. It further suggests that correcting this HO-1 deficiency in HIV-infected MDM could provide neuroprotection above that provided by current ART or proposed antiviral therapies directed at limiting Nef, Vpr or Vpu functions. The ability of HIV-2 to reduce HO-1 expression suggests that this is a conserved phenotype among macrophage-tropic human immunodeficiency viruses that could contribute to neuropathogenesis.

    Importance The continued prevalence of HIV-associated neurocognitive disorders (HAND) underscores the need for adjunctive therapy that targets the neuropathological processes that persist in antiretroviral therapy (ART)-treated HIV-infected individuals. To this end, we previously identified once such possible process, a deficiency of the anti-oxidative and anti-inflammatory enzyme heme oxygenase-1 (HO-1) in the brains of individuals with HAND. In our current study, our findings suggest that the HO-1 deficiency associated with excess glutamate production and neurotoxicity in HIV-infected macrophages is a highly conserved phenotype of macrophage-tropic HIV strains, and that this phenotype can persist in the macrophage compartment in the presence of ART. This suggests a plausible mechanism by which HIV infection of brain macrophages in ART-treated individuals could exacerbate oxidative stress and glutamate-induced neuronal injury, each of which is associated with neurocognitive dysfunction in infected individuals. Thus, therapies that rescue the HO-1 deficiency in HIV-infected individuals could provide additional neuroprotection to ART.

  • ENCAPSIDATION OF HOST RNAS BY CUCUMBER NECROSIS VIRUS COAT PROTEIN BOTH DURING AGRO-INFILTRATION AND INFECTION [PublishAheadOfPrint]

  • Next-generation sequence analysis of virus like particles (VLPs) produced during agroinfiltration of Cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions has been conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60th the level that CNV virions accumulate during infection. VLPs contained a variety of host RNA species including the major rRNAs as well as cytoplasmic, chloroplast and mitochondrial mRNA. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that CNV CP expressed during agroinfiltration was the most efficiently encapsidated mRNA suggesting that the CNV CP ORF may contain a high affinity site or sites for CP binding and thus contribute to the specificity of CNV viral RNA encapsidation. Approximately 0.09% nndash; 0.7% of RNA derived from authentic CNV virions contained host RNA, again with chloroplast RNA being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6-8 of the top ten most efficiently encapsidated nuclear encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution.

    IMPORTANCE Viruses predominantly encapsidate their own viral-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high affinity binding sites for the coat protein. In this study we show using next-generation sequence analysis that CNV also encapsidates host RNA species, which account for ~0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs include predominantly chloroplast RNAs reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences similar to that recently reported for Flock house virus (A. Routh, T. Domitrovic and J.E. Johnson, PNAS 109: 1907-1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large scale genomic changes in a naiiuml;ve plant host, thus facilitating host evolutionary novelty.

  • Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-like Growth Factor Receptors to Mediate Constitutive Myc Expression [PublishAheadOfPrint]

  • The E4-ORF1 protein encoded by human adenovirus stimulates viral replication in human epithelial cells by binding and activating cellular phosphatidylinositol 3-kinase (PI3K) at the plasma membrane and cellular Myc in the nucleus. In the current study, we showed that E4-ORF1 hijacks the tyrosine kinase activities of cellular epidermal growth factor receptor (EGFR) and insulin receptor (InsR)/insulin-like growth factor receptor 1 (IGF1R), as well as the lipid kinase activity of PI3K, to mediate constitutive Myc protein expression. We additionally demonstrated that EGFR contributes to constitutive Myc expression through the capacity of E4-ORF1 to induce ligand-independent EGFR activation and stimulation of the Ras/Mek/Erk pathway, the latter activity of which was conserved by human adenoviruses. Results further suggested that EGFR normally forms a complex with the cellular PDZ protein Discs Large 1 (Dlg1), a component of the Dlg1:E4-ORF1:PI3K ternary complex that mediates E4-ORF1-induced PI3K activation, and that E4-ORF1 binds the Dlg1:EGFR complex and promotes the association of EGFR with InsR and IGF1R. In addition to its role in constitutive Myc expression, InsR/IGF1R also negatively regulated EGFR autophosphorylation and EGFR-mediated Ras/Mek/Erk signaling, and data suggested that E4-ORF1 binding to Dlg1 antagonizes these activities. Collectively, our findings suggest that in human epithelial cells, E4-ORF1 targets EGFR, InsR/IGF1R, and PI3K at the plasma membrane to activate cytosolic signaling pathways that sustain Myc protein levels in the nucleus. We postulate that E4-ORF1-induced constitutive Myc expression functions to ensure the formation of nuclear E4-ORF1:Myc complexes, which have been shown to activate Myc and to enhance adenovirus replication.

    IMPORTANCE While human adenoviruses primarily produce self-limited acute infections in humans, these agents are associated with life-threatening diseases in immune-compromised patients and in otherwise healthy individuals infected with certain virulent serotypes. The adenovirus E4-ORF1 protein enhances viral replication by activating the cellular lipid kinase PI3K and cellular transcription factor Myc. Here we report that E4-ORF1 usurps the functions of the cellular tyrosine kinase receptors EGFR and InsR/IGF1R, as well as PI3K, to sustain Myc protein expression in cells. Further, sustained Myc expression depended on E4-ORF1-induced ligand-independent EGFR activation that stimulated Ras/Mek/Erk signaling, a function found to be conserved by human adenoviruses. Given the established roles of PI3K, the Ras/Mek/Erk pathway, and Myc in the adenovirus life cycle, our findings may aid development of safer, more effective therapeutic strategies to treat severe adenovirus infections, as well as improved adenovirus vectors for use in vaccination and gene and cancer therapy.

  • Generation of live attenuated influenza virus using codon usage bias [PublishAheadOfPrint]

  • Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses of vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells, but not in eggs, by converting its codon usage similar to those observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus our approach is a successful strategy to generate attenuated viruses for future vaccine application.

    Importance Vaccination has been one of the best protective measures in combating influenza infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect the virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern of reverse mutations. The potential use of this kind of codon bias mutants as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.

  • Increased immune response variability during simultaneous viral co-infection leads to unpredictability in CD8 T cell immunity and pathogenesis1 [PublishAheadOfPrint]

  • T cell memory is usually studied in the context of infection with a single pathogen in naiiuml;ve mice, but how memory develops during a co-infection with two pathogens, as frequently occurs in nature or after vaccination, is far less studied. Here, we questioned how the competition between immune responses to two viruses in the same naiiuml;ve host would influence the development of CD8 T cell memory and subsequent disease outcome upon challenge. Using two different models of co-infection, including the well-studied lymphocytic choriomeningitis (LCMV) and Pichinde (PICV) viruses, several differences were observed within the CD8 T cell responses to either virus. Compared to single-virus infection, co-infection resulted in substantial variation among mice in the size of epitope-specific T cell responses to each virus. Some mice had an overall reduced number of virus-specific cells to either one of the viruses and others developed an immunodominant response to a normally subdominant, crossreactive epitope. These changes led to decreased protective immunity and enhanced pathology in some mice upon challenge with either of the original co-infecting viruses. In mice with PICV-dominant responses, during a high dose challenge with LCMV clone 13, increased immunopathology was associated with the reduced number of LCMV-specific effector memory CD8 T cells. In mice with dominant crossreactive memory responses, during challenge with PICV, increased immunopathology was directly associated with these crossreactive NP205-specific CD8 memory cells. In conclusion, the inherent competition between two simultaneous immune responses results in significant alternations in T cell immunity and subsequent disease outcome upon re-exposure.

    IMPORTANCE Combination vaccines and simultaneous administration of vaccines are necessary to accommodate required immunizations and maintain vaccination rates. Antibody responses generally correlate with protection and vaccine efficacy. However, live-attenuated vaccines also induce strong CD8 T-cell responses, and the impact of these cells on subsequent immunity, whether beneficial or detrimental, has seldom been studied, in part due to the lack of known T-cell epitopes to vaccine viruses. We questioned if the inherent increased competition and stochasticity between two immune responses during a simultaneous co-infection would significantly alter CD8 T-cell memory in a mouse model, where CD8 T-cell epitopes are clearly defined. We show that some of the co-infected mice have sufficiently altered memory T-cell responses that they have decreased protection and enhanced immunopathology when re-exposed to one of the two viruses. These data suggest that a better understanding of human T-cell responses to vaccines is needed to optimize immunization strategies.

  • Dead box helicase 60-like (DDX60L) is an interferon stimulated gene restricting hepatitis C virus replication in cell culture [PublishAheadOfPrint]

  • All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 to identify effector genes of the IFN response and thereby identified the DExD/H box helicase DDX60L as a restriction factor of HCV replication. DDX60L and its homolog DDX60 were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells, and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was only detectable upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon stimulated gene induction after IFN treatment, but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found no impact of DDX60L on translation or stability of HCV subgenomic replicons, nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity suggesting additional functions in the sensing of viral RNA.

    Importance Interferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV), still their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biology. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

  • Flexibility of NS5 Methyltransferase-Polymerase Linker Region is Essential for Dengue virus Replication [PublishAheadOfPrint]

  • We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263-272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.

  • Structures of coxsackievirus A16 capsids with native antigenicity, implications for particle expansion, receptor binding and immunogenicity [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) which affect more than a million children in China each year, and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess non-native antigenicity are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. We here remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant virus-like particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilise the native antigenic state that is different to that used in a natural virus infection. As expected the mature CVA16 virus is similar to EV71, however structural and immunogenic comparisons highlight differences, which may have implications for vaccine production.

    IMPORTANCE Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of a disease that, whilst usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, natural empty particles, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles produced in insect cells (a system suitable for making vaccine antigen) are stabilised by recruiting from the insect cells a small molecule different to that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug-design and vaccine development.

  • SARS coronavirus ORF8 protein is acquired from SARS-related coronavirus from greater horseshoe bats through recombination [PublishAheadOfPrint]

  • Despite the identification of horseshoe bats as the reservoir of SARS-related-coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nt signature deletion among human strains, remains obscure. Although two SARSr-Rs-BatCoVs, RsSHC014 and Rs3367, previously detected from Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, possessed 95% genome identities to human/civet SARSr-CoVs, their ORF8 exhibited only 32.2-33% aa identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nt identities to human/civet SARSr-CoV genomes. Although they displayed lower similarities to civet SARSr-CoVs than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein, their ORF8 demonstrated exceptionally high (80.4-81.3%) aa identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2-37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that this protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSs-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs at approximately 1990. SARS-CoV ORF8 is originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission.

    IMPORTANCE Although horseshoe bats are the primary reservoir of SARS-related-coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civet/human. Most human SARS-CoV epidemic strains contained a signature 29-nt deletion in ORF8 compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats exhibited llt;40% aa identities to human/civet SARS-CoV in ORF8. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed an ORF8 with exceptionally high aa identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination.

  • Inhibition of histone deacetylase activity aggravates coxsackievirus B3-induced myocarditis by promoting viral replication and myocardial apoptosis [PublishAheadOfPrint]

  • Viral myocarditis, which is most prevalently caused by coxsackievirus B3 (CVB3), is a serious clinical condition characterized by excessive myocardial inflammation. Recent studies suggest that regulation of protein acetylation levels by inhibiting histone deacetylase (HDAC) activity modulates inflammatory response and shows promise as a therapy for several inflammatory diseases. However, the role of HDAC activity in viral myocarditis is still not fully understood. Here, we aim to investigate the role of HDAC activity in viral myocarditis and its underlying mechanism. CVB3-infected BALB/c mice were treated with HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA). We found inhibition of HDAC activity aggravated rather than ameliorated the severity of CVB3-induced myocarditis, which was contrary to our expectations. The aggravated myocarditis by HDACI treatment seemed not to be caused by an elevated inflammatory response, but by the increased CVB3 replication. Further, it was revealed that the increased CVB3 replication was closely associated with the HDACI-enhanced autophagosome formation. Inhibition of autophagosome formation by wortmannin or ATG5 shRNA dramatically suppressed the HDACI-increased CVB3 replication. The increased viral replication subsequently elevated CVB3-induced myocardial apoptosis. Conversely, inhibition of CVB3 replication and ensuing myocardial apoptosis by antiviral drug ribavirin significantly reversed the HDACI-aggravated viral myocarditis. In conclusion, we elucidate that the inhibition of HDAC activity increases CVB3 replication and ensuing myocardial apoptosis, resulting in aggravated viral myocarditis. Possible adverse consequences of administering HDACI should be considered in patients infected (or co-infected) with CVB3.

    IMPORTANCE Viral myocarditis, which is most prevalently caused by coxsackievirus B3 (CVB3), is characterized by excessive myocardial inflammation. Inhibition of HDAC activity was originally identified as a powerful anti-cancer therapeutic strategy and was recently found to be implicated in the regulation of inflammatory response. HDAC inhibitor (HDACI) is demonstrated efficacious in animal models of several inflammatory diseases. We thus hypothesize that inhibition of HDAC activity also protects against CVB3-induced viral myocarditis. Surprisingly, we found inhibition of HDAC activity enhanced myocardial autophagosome formation, which led to the elevated CVB3 viral replication and ensuing increased myocardial apoptosis. Viral myocarditis was eventually aggravated rather than ameliorated by HDAC inhibition. In conclusion, we elucidate the role of HDAC activity in viral myocarditis. Moreover, given the importance of HDACI in preclinical and clinical treatments, the possible unfavorable effect of HDACI should be carefully evaluated in patients infected with viruses, including CVB3.

  • Intermonomer Interactions in Hemagglutinin subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity [PublishAheadOfPrint]

  • Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop) to helix transition, which releases the fusion peptide at the HA2 N-terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable relative to other seasonal influenza strains. Here we show that residue 205 in HA1 and 399 in the B loop of HA2 (residue 72, HA2 number) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity.

    IMPORTANCE Influenza hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, influenza A HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity, but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza HA, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.

  • Suppressor of cytokine signaling 3 is an inducible host factor during Ebola virus infection that regulates virus egress [PublishAheadOfPrint]

  • Ebola virus (EBOV) initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal EBOV infections. The VP40 matrix protein is a key viral structural protein that is critical for virion egress. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VP40-driven virus-like particles (VLPs) and infectious virus. Here, we show that host suppressor of cytokine signaling 3 (SOCS3) can also bind to EBOV VP40, leading to enhanced ubiquitinylation and egress of VP40. Indeed, titers of infectious EBOV derived from SOCS3 knockout MEFs were significantly reduced compared to those from WT MEFs at 24 and 48 hours post-infection. Importantly, this reduced virus yield could be rescued back to WT levels by exogenously expressing SOCS3. Lastly, we show that SOCS3 expression is induced by EBOV glycoprotein (GP) expression, and that VLPs containing EBOV VP40 and GP induced production of proinflammatory cytokines, which induced SOCS3 for negative feedback regulation. These data indicate that host innate immune protein SOCS3 may play an important role in budding and pathogenesis of EBOV.

    Importance The VP40 matrix protein is a key structural protein critical for Ebola virus budding. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VLPs and infectious virus. We reported that host TLR4 is a sensor for Ebola GP on VLPs, and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines. Host SOCS3 regulates the innate immune response by controlling and limiting the proinflammatory response through negative-feedback inhibition of cytokine receptors. We present evidence that Ebola virus VLPs stimulate induction of SOCS3 as well as proinflammatory cytokines, and that expression of human SOCS3 enhances budding of Ebola VLPs and infectious virus via a mechanism linked to the host ubiquitinylation machinery.

  • Dolutegravir-selected HIV-1 containing the N155H/R263K resistance substitutions does not acquire additional compensatory mutations under drug pressure that lead to higher level resistance and increased replicative capacity [PublishAheadOfPrint]

  • We have previously shown that the addition of the raltegravir/elvitegavir (RAL/EVG) primary resistance mutation N155H to the R263K dolutegravir (DTG) resistance mutation partially compensated for the fitness cost imposed by R263K while also slightly increasing DTG resistance in vitro. Since many patients failing RAL/EVG are given DTG as part of rescue therapy, and given that the N155H substitution is often found in combination with other compensatory resistance mutations in such individuals, we investigated the effects of multiple such substitutions within integrase (IN) on each of integrase function, HIV-1 infectivity, and levels of drug resistance. To this end, each of the L74M, E92Q, T97A, E157Q, and G163R substitutions were introduced into NL4.3 subtype B HIV-1 vectors harboring N155H and R263K in tandem. Relevant recombinant integrase enzymes were also expressed and purified and biochemical assays of strand-transfer efficiency as well as viral infectivity and drug resistance studies were performed. We found that the addition of T97A, E157Q, or G163R somewhat improved the affinity of INN155H/R263K for its target DNA substrate while the presence of L74M or E92Q had a negative effect on this process. However, viral infectivity was significantly decreased from that of NL4.3IN(N155H/R263K) after the addition of each tertiary mutation and no increases in levels of DTG resistance were observed. This work shows that the compensatory mutations that evolve after N155H under continued DTG or RAL/EVG pressure in patients are unable to improve either enzyme efficiency or viral infectivity in a N155H/R263K background.

    Importance: In contrast to other drugs, dolutegravir has not selected for resistance in HIV-positive individuals when used in first line therapy. We had previously shown that HIV containing the primary raltegravir/elvitegravir resistance substitution N155H could select for R263K under dolutegravir pressure and that this virus was fit and displayed low-level resistance to dolutegravir. Therefore, the current study aimed to uncover whether accessory mutations that appear after N155H in response to raltegravir/elvitegravir were compatible with N155H and R263K. We found, however, that the addition of a third mutation negatively impacted both the enzyme and the virus in terms of activity and infectivity without large shifts in integrase inhibitor resistance. Thus, it is unlikely that these substitutions would be selected under dolutegravir pressure. These data support the hypothesis that primary resistance against DTG cannot evolve through RAL/EVG resistance pathways and that the selection of R263K leads HIV into an evolutionary dead-end.

  • Structural and functional diversity of nairovirus-encoded nucleoproteins [PublishAheadOfPrint]

  • The nairoviruses include assorted tick-borne bunyaviruses that are emerging as causative infectious diseases among humans and animals. As negative-sense single-stranded RNA (-ssRNA) viruses, nairoviruses encode nucleoprotein (NP) that encapsidates the genomic RNA and further forms ribonucleoprotein complex (RNP) with viral RNA-dependent RNA polymerase (RdRp). We previously revealed that the monomeric NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV) presents a racket-shaped structure and shows unusual DNA-specific endonuclease activity. To examine the structural and biological variation of nairovirus-encoded NPs, here we systematically solved the crystal structures of NPs encoded by various nairoviruses, including Hazara virus (HAZV), Kupe virus (KUPV), and Erve virus (ERVEV). Combined with biochemical analysis, our results generate a clearer picture to aid in the understanding of the functional diversity of nairovirus-encoded NPs and the formation of nairovirus RNPs.

    Importance Nairoviruses are consisted of several tick-borne bunyaviruses that are emerging as causative infectious diseases among humans and animals, however, little is known of the nairovirus genome assembling and transcription mechanism. Based on the previous study of CCHFV NP reported by different research groups, here we systematically investigate the structural and functional diversity among three different Nairoviruses, these work provide important information on nairovirus nucleoprotein function and the formation of RNPs.

  • Murine antibody responses to cleaved soluble HIV-1 envelope trimers are highly restricted in specificity [PublishAheadOfPrint]

  • Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive, and thus capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and are likely occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it may be useful to obscure non-neutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs).

    IMPORTANCE Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies could not induce neutralizing antibodies to a neutralization-resistant HIV strain. Further analysis revealed that mouse antibodies targeted areas near the bottom of the soluble envelope trimers. These areas are not easily accessible on the HIV virion due to occlusion by the viral membrane and may have resulted from an absence of a glycan shielding. Our results suggest that obscuring the bottom of soluble envelope trimers may be useful strategy to reduce antibody responses to epitopes that are not useful for virus neutralization.

  • Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs [PublishAheadOfPrint]

  • CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope glycoproteins (Env) on these HIV-1 expose coreceptor binding site, which overlaps with some CD4-induced (CD4i) epitopes. Reports have demonstrated CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140(-)) from a CD4-independent HIV-1 in guinea pigs and found the sgp140(-) elicited ADCC-mediating antibodies. Therefore, these sgp140(-) might be useful in vaccine regiments aimed at eliciting ADCC responses.

  • A role for a zinc finger protein (Zfp111) in transformation of 208F rat fibroblasts by Jaagsiekte Sheep Retrovirus envelope protein. [PublishAheadOfPrint]

  • The native envelope gene (env) of Jaagsiekte Sheep Retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation we performed yeast 2-hybrid screening for cellular proteins that interact with Env.Among several candidates, we identified mouse or rat Zinc Finger Protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo co-immunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Envwhile over-expression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111.In addition, Zfp111 preferentially bound to a higher mobility form of JSRV Env that has not been described previously. The higher mobility form of Env (P70env) was found exclusively in the nuclear fraction, and its polypeptide backbone was the same size as that of cytoplasmic Env polyprotein (Pr80env). The differences in glycosylation between the two versions of Env were characterized.These results identify a novel cellular protein Zfp111 that binds to JSRV Env protein, and this binding plays a role in Env transformation. They indicate that JSRV transformation also involves proteins and interactions in the nucleus.

    IMPORTANCE The envelope protein (Env) by Jaagsiekte Sheep Retrovirus (JSRV) is an oncogene, but itsmechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, Zinc Finger Protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells, and to be involved in JSRV transformation. Moreover co-expression of JSRV Env and Zfp111 identified a novel nuclear form of JSRV Env protein that binds Zfp111. Nuclear Env was found to differ by glycosylation from the cytoplasmic Env precursor to the virion envelope proteins. These results suggest that JSRV Env transformation may involve nuclear events such as alteration in transcription mediated by Env-Zfp111 interactions.

  • Hydrodynamic and membrane binding properties of purified Rous sarcoma virus Gag protein [PublishAheadOfPrint]

  • Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein, and in parallel several truncation mutants of Gag, and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag which is compact. However, by neutron reflectometery measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of PI(4,5)P2 and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. By contrast with full length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain, and a C-terminally truncated version that is missing the NC domain, bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    IMPORTANCE Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a "folded-over" conformation on a lipid bilayer implicating both the N- and C-terminus in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral lifecycle, and an untapped target for anti-retroviral drugs.

  • A druggable pocket at the nucleocapsid/phosphoprotein interaction site of the human respiratory syncytial virus [PublishAheadOfPrint]

  • Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently by antivirals. However, its RNA dependent polymerase complex offers potential targets for RSV specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein N. This recognition proceeds via interaction between the phosphoprotein P, that is the main polymerase co-factor, and N. The determinant role of the C-terminus of P and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here we provide a detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structure of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside of the pocket. Based on the structural information of the N-NTD:P complex we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV specific antivirals.

    Importance Respiratory Syncytial Virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently by antivirals and no vaccine is presently available, the development of pediatric vaccines being particularly challenging. Therefore there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study we identified the main structural determinants of this interaction, and we used them to in silico screen potential inhibitors. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a base for a pharmacophore model that must be improved, but holds promises for the design of new RSV specific antivirals.

  • Topography of the human papillomavirus minor capsid protein L2 during vesicular trafficking of infectious entry [PublishAheadOfPrint]

  • The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if by direct interaction, would interact with cytosolic host cell factors to facilitate this transport. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64-81 and 163-170 and a C-terminal tag of 16L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20-38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking.

    IMPORTANCE In order to complete infectious entry, non-enveloped viruses have to pass cellular membranes. This is often achieved through viral capsid protein associating with or integrating into intracellular membrane. Herein, we determine the topography of HPV L2 protein in the endocytic vesicular compartment suggesting that L2 becomes a transmembrane protein with a short luminal portion and the majority facing the cytosolic side for interaction with host cell transport factors.

  • Host determinants of prion strain diversity independent of prion protein genotype [PublishAheadOfPrint]

  • Phenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrPSc conformations. Here we investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but following transmission to SGH, the incubation period of CKY CWD was ~150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrPSc polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrPSc differed for the CWD strains in a host-dependent manner. WST CWD produced PrPSc amyloid plaques in the brain of SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrPSc from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrPSc properties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent of Prnp can alter the PrPSc conformation of certain prion strains, leading to changes in the biophysical properties of PrPSc, neuropathology, and clinical prion disease.

    IMPORTANCE Prions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the current study we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

  • Establishment of a zebrafish infection model for the study of wild type and recombinant European sheatfish virus [PublishAheadOfPrint]

  • Amphibian-like ranaviruses include pathogens of fish, amphibians and reptiles that have recently evolved from a fish-infecting ancestor. The molecular determinants of host range and virulence in this group are largely unknown and currently fish infection models are lacking. We show that European sheatfish virus (ESV) can productively infect zebrafish causing a lethal pathology and describe a method for the generation of recombinant ESV establishing a useful model for the study of fish ranavirus infections.

  • Turnover rate of NS3 proteins modulates bluetongue virus replication kinetics in a host-specific manner [PublishAheadOfPrint]

  • Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family, and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are primarily released by virus-induced cell lysis while, in insect cells, they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double stranded RNA genome and NS3 proteins are encoded by segment 10 (Seg-10). The viral non-structural protein 3 (NS3) plays a key role in mediating BTV egress, as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-hosts interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells, and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner, and highlight the role of the host-specific variation in NS3 proteins turnover rate.

    Importance Bluetongue virus (BTV) is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. The non-structural protein 3 (NS3) encoded by the segment 10 (Seg-10) of BTV genome fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and hosts interactions. In this study, we revealed that various Seg-10/NS3 alter BTV replication kinetics in mammals but not in insects. Notably, we found that the NS3 proteins turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner, and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.

  • Constitutive activation of IL-13/STAT6 contributes to KSHV-associated PEL cell proliferation and survival [PublishAheadOfPrint]

  • Activation of the Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway has been associated with numerous human malignancies, including primary effusion lymphomas (PELs). PEL, a cancerous proliferation of B-cells, is caused by the Kaposi's sarcoma-associated herpesvirus (KSHV). Previously we identified constitutive phosphorylation of STAT6 on tyrosine 641 (p-STAT6c) in PEL cell lines BC3 and BCBL1, however, the molecular mechanism leading to this activation remains unclear. Here we demonstrate that STAT6 activation tightly correlates with interleukin-13 (IL-13) secretion, JAK1/2 tyrosine phosphorylation, and reduced expression of SHP1 due to KSHV infection. Moreover, p-STAT6c and reduction of SHP1 were also observed in KS patient tissue. Notably, blockade of IL-13 by antibody neutralization dramatically inhibits PEL cell proliferation and survival. Taken together, these results suggest that IL-13/STAT6 signaling is modulated by KSHV to promote host cell proliferation and viral pathogenesis.

    IMPORTANCE STAT6 is a member of signal transducer and activator of transcription (STAT) family, whose activation is linked to KSHV-associated cancers. The mechanism through which STAT6 is modulated by KSHV remains unclear. In this study, we demonstrate that constitutive activation of STAT6 in KSHV-associated PEL cells, results from interleukin-13 (IL-13) secretion and reduced expression of SHP1. Importantly, we also found that depletion of IL-13 reduces PEL cell growth and survival. This discovery provides a new insight that IL-13/STAT6 plays an essential role in KSHV pathogenesis.

  • Importation and recombination are responsible for the latest emergence of highly pathogenic PRRSV in China [PublishAheadOfPrint]

  • In China, majority of the highly pathogenic PRRSV (HP-PRRSV) are seeded by the 2006 outbreak. However, the most recently emerged (2013-2014) HP-PRRSV has a very different genetic background. It is a NADC30-like HP-PRRSV recently introduced from North America and has undergone genetic exchange with the classic HP-PRRSVs in China. Subsequent isolation and characterization of this variant suggests high pathogenicity, so it merits special attention on control and vaccine strategies.

  • Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity [PublishAheadOfPrint]

  • We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon-antagonists, but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA-binding required conserved basic residues in this domain. Interferon-antagonism was strictly dependent on RNA-binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells, but not in cells unable to establish antiviral immunity.

  • Neuraminidase of influenza A virus binds LAMPs directly and induces lysosome rupture [PublishAheadOfPrint]

  • As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently lysosomal cell death has been defined as "lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol". Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosomal-associated membrane proteins (LAMPs, the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells (HTEpiC). The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle, but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death.

    Importance The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosomal-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function as well as for influenza inhibitors' mechanism of action and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings, and why NA inhibitors have more potent therapeutic effects for lethal avian influenza infections at early stage.

  • A global database-driven assessment of HIV-1 adaptation to the immune repertoires of human populations [PublishAheadOfPrint]

  • Associations between HIV-1 CTL escape mutations and their restricting HLA alleles imply that HIV could adapt to divergent HLA repertoires of human populations globally. Using publicly-available databases, we examine the relationship between the frequencies of 19 experimentally-validated CTL escape mutations in HIV-1 reverse transcriptase and their restricting HLA alleles in 59 countries. From these extensive data, we find evidence of differential HIV adaptation to human populations at only a limited number of studied epitope sites.

  • Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site [PublishAheadOfPrint]

  • Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study we generated a reassortant H5N3 virus that possessed the HA gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3 inoculated birds replicated and shed virus and seroconverted, transmission to naiiuml;ve contacts did not occur. To determine if this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole genome sequencing demonstrated the fixation of twelve non-synonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low pathogenic rH5N3 increased its virulence, transmission to naiiuml;ve contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype.

    IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits like antiviral drug resistance.

  • Identification of host cell factors associated with astrovirus replication in Caco-2 cells [PublishAheadOfPrint]

  • Astroviruses are small, non-enveloped viruses with a single-stranded positive-sense RNA genome, causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have been frequently found to replicate associated to membranous structures, in this work we characterized the replication of the human astroviruses serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles, were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells, were identified by tandem mass spectrometry. Results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIII-bbeta;) and inositol-phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23), significantly decreased amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles.

    IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work we provide evidence that astrovirus RNA replication and virus assembly occurs in contact with cell membranes potentially derived from multiple cell organelles and show that cellular proteins involved in lipid metabolism that associate with these membranes are required for the efficient viral replication. Our findings provide information to the knowledge of astrovirus biology and provide information that might be useful to develop therapeutic interventions to prevent virus replication.

  • Virus-Like Vesicle-Based Therapeutic Vaccine Vectors for Chronic Hepatitis B Virus Infection [PublishAheadOfPrint]

  • More than 500,000 people die each year from the liver diseases that result from chronic hepatitis B virus (HBV) infection. Therapeutic vaccines, which aim to elicit an immune response capable of controlling the virus, offer a potential new treatment strategy for chronic hepatitis B. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the vesicular stomatitis virus glycoprotein (VSV G) has been described. This platform generates "virus-like vesicles" (VLVs) that contain VSV G but no other viral structural proteins. We report here that the evolved VLV vector engineered to additionally express the HBV middle surface envelope glycoprotein (MHBs) induces functional CD8 T cell responses in mice. These responses were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. Additionally, a single immunization with VLV-MHBs protected mice from HBV hydrodynamic challenge, and this protection correlated with the elicitation of a CD8 T cell recall response. In contrast to MHBs, a VLV expressing HBV Core protein (HBcAg) neither induced a CD8 T cell response in mice, nor protected against challenge. Finally, combining DNA and VLV-MHBs immunization led to induction of HBV-specific CD8 T cell responses in a transgenic mouse model of chronic HBV infection. The ability of VLV-MHBs to induce a multispecific T cell response capable of controlling HBV replication, and to generate immune responses in a tolerogenic model of chronic infection, indicates that VLV vaccine platforms may offer a unique strategy for HBV therapeutic vaccination.

    IMPORTANCE HBV infection is associated with significant morbidity and mortality. Furthermore, treatments for chronic infection are suboptimal and rarely result in complete elimination of the virus. Therapeutic vaccines represent a unique approach to HBV treatment and have the potential to induce long-term control of infection. Recently, a virus-based vector system that combines the nonstructural proteins of Semliki Forest virus with the VSV glycoprotein has been described. In this study, we use this system to construct a novel HBV vaccine and demonstrate that the vaccine is capable of inducing virus-specific immune responses in mouse models of acute and chronic HBV replication. These findings highlight the potential of this new vaccine system and support the idea that highly immunogenic vaccines, such as viral vectors, may be useful in the treatment of chronic hepatitis B.

  • Hepatitis C virus envelope glycoprotein E1 forms trimers at the surface of the virion [PublishAheadOfPrint]

  • In hepatitis C virus (HCV) infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the co-expression of E2. To further understand the origin of E1 trimer formation, we co-expressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homo-dimers and -trimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif which is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as the homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles and support the hypothesis that E1 could be a fusion protein.

    Importance HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the fusion process between viral and cellular membranes. Here, we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement of E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

  • Evasion of the innate immune type I interferon system by monkeypox virus [PublishAheadOfPrint]

  • The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing for viral replication to occur unhindered. Mutations or deletions of E3L severely affect viral host range and pathogenesis. While monkeypox virus (MPXV) encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein truncated of 37 N-terminal amino acids. VACV encoding a similarly truncated E3 protein (VACV-E3L37N) has been shown to be attenuated in mouse models, and to lead to activation of the host anti-viral PKR pathway. In this manuscript we present data demonstrating that, despite containing a truncated E3-homologue, MPXV phenotypically resembles a wild type VACV (wt-VACV), rather than VACV-E3L37N. Thus, MPXV appears to contain a gene or genes that can suppress phenotypes associated with an N-terminal truncation in E3. Suppression maps to sequences outside of F3L, suggesting that the suppression is extra-genic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue.

    IMPORTANCE Poxviruses have evolved to have many mechanisms to evade host anti-viral innate immunity; these mechanisms may allow these viruses to cause disease. Within the family of poxviruses, Variola (smallpox) is the most pathogenic, while monkeypox virus is intermediate in pathogenicity between vaccinia virus and Variola. Understanding the mechanisms of monkeypox virus innate-immune evasion will help us to understand the evolution of poxvirus innate-immune evasion capabilities, providing a better understanding of how poxviruses cause disease.

  • Fitness-balanced escape determines resolution of dynamic founder virus escape processes in HIV-1 infection [PublishAheadOfPrint]

  • To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days post infection. T-cell responses were screened by IFN/IL-2 FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r=0.43 p=0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack.

    Importance Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host, and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens.

  • R5 Macrophage-Tropic HIV-1 in the Male Genital Tract [PublishAheadOfPrint]

  • The entry tropism of HIV-1 Env proteins from virus isolated from the blood and genital tract of five men with compartmentalized lineages was determined. The Env proteins isolated from the genital tract of subject C018 were macrophage-tropic, while the remaining cloned env genes were R5 T cell-tropic. The detection of a macrophage-tropic lineage of HIV-1 within the male genital tract strongly suggests that evolution of macrophage-tropic viruses can occur in anatomically isolated sites outside of the central nervous system.

  • Inhibition of HIV expression and integration in macrophages by Methylglyoxal-bis-guanylhydrazone [PublishAheadOfPrint]

  • Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current anti-retroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages.

    IMPORTANCE Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage targeted therapy for HIV infection.

  • RIG-I mediates an antiviral response to Crimean-Congo hemorrhagic fever virus [PublishAheadOfPrint]

  • In the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative sense RNA virus with a 5rrsquo; -monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health and clinical implications. We found that, during CCHFV infection, RIG-I mediated a type-I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression, and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I co-immunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV.

    IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus with significant public health and clinical impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in recognition of CCHFV are not known. Herein we investigate and identify retinoic acid-inducible gene I (RIG-I) as a receptor involved in initiating an antiviral response to CCHFV. This receptor was initially not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I.

  • Bub1 in complex with LANA recruits PCNA to regulate KSHV latent replication and DNA translesion synthesis [PublishAheadOfPrint]

  • Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs and LANA. However, how cellular proteins are recruited to the viral genome is not very clear. Here we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with PCNA via a highly conserved PIP-box motif within the Kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV positive cells. This strongly indicated that Bub1 may serve as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S-phase and interacted with PCNA to promote its mono-ubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV infected cells.

    IMPORTANCE In KSHV latency infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV latency-associated nuclear antigen (LANA) recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with proliferating cell nuclear antigen (PCNA), a critical protein for viral DNA replication. Furthermore our findings suggest that Bub1, a spindle checkpoint protein, may serve as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as mono-ubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

  • Human dendritic cell response signatures distinguish 1918, pandemic and seasonal H1N1 influenza viruses [PublishAheadOfPrint]

  • Influenza viruses continue to present global threats to human health. Antigenic drift and shift, genetic reassortment, and cross-species transmission generate new strains with differences in epidemiology and clinical severity. We compared the temporal transcriptional responses of human dendritic cells (DC) to infection with two pandemic (A/Brevig Mission/1/1918, A/California/4/09) and two seasonal (A/New Caledonia/20/99, A/Texas/36/1991) H1N1 influenza viruses. Strain-specific response differences included stronger activation of NFB following infection with A/New Caledonia/20/99 and a unique cluster of genes expressed following infection with A/Brevig Mission/1/1918. A common anti-viral program showing strain-specific timing was identified in the early DC response and found to correspond with reported transcript changes in blood during symptomatic human influenza infection. Comparison of the global response to the seasonal and pandemic strains showed that a dramatic divergence occurred after 4 h, with only the seasonal strains inducing widespread mRNA loss.

    Importance Continuously evolving influenza viruses present a global threat to human health however, these host responses display strain-dependent differences that are incompletely understood. Thus we conducted a detailed comparative study comparing the immune response of human DC to infection with two pandemic and two seasonal H1N1 influenza strains. We identified in the immune response to viral infection both common, but also strain specific, features. Among the stain specific elements were a time shift of the ISG response, selective induction of NFB signaling by one of the seasonal strains and massive RNA degradation as early as 4 hours post-infection by the seasonal, but not the pandemic, viruses. These findings illuminate new aspects that characterize the distinct differences in the immune response to pandemic or seasonal influenza viruses.

  • The structure of immature-like Rous sarcoma virus Gag particles reveals a structural role for the p10 domain in assembly [PublishAheadOfPrint]

  • The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved CA domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here we have employed cryo-electron tomography and subtomogram averaging to study in vitro assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles, and have determined the structure of CA and the surrounding regions to a resolution of ~ 8 AAring;. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, while the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, consistent with structural regulation of proteolytic maturation.

    IMPORTANCE Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. Here we have used techniques called cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We find that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

  • Innate immune activity correlates with CD4 T cell-associated HIV-1 DNA decline during latency-reversing treatment with panobinostat [PublishAheadOfPrint]

  • Pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1 infected cells in individuals treated with suppressive antiretroviral combination therapy (cART). However, effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1 patients. We observed that the magnitude, breadth, and cytokine-secretion profile of HIV-1-specific CD8 T cell responses were unrelated to HIV-1 DNA changes in CD4 T cells during panobinostat treatment. In contrast, proportions of CD3- CD56+ total NK cells and CD16+ CD56dim NK cells were inversely correlated with HIV-1 DNA throughout the study, and changes of HIV-1 DNA during panobinostat treatment were negatively associated with corresponding changes in CD69+ NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to proportions of plasmacytoid dendritic cells, to distinct gene expression patterns of interferon-stimulated genes and to expression of the IL28B "CC" genotype. Together, these data suggest that innate immune activity can critically modulate effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies.

    IMPORTANCE Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists despite treatment in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed as the "shock and kill" strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial, and reduce HIV-1 DNA in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells and expression patterns of Interferon-stimulated genes, were most closely linked to a decline of HIV-1 DNA during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1 infected cells.

  • Pathogenesis and transmission of novel HPAI H5N2 and H5N8 avian influenza viruses in ferrets and mice [PublishAheadOfPrint]

  • A novel highly pathogenic avian influenza (HPAI) H5N8 virus, first detected in January 2014 in poultry and wild birds in South Korea, has spread throughout Asia and Europe, and caused outbreaks in Canada and the United States by the end of the year. The spread of H5N8 and the novel reassortant viruses, H5N2 and H5N1 (H5Nx), in domestic poultry across multiple states in the U.S. pose a potential public health risk. To evaluate the potential of cross-species infection, we determined the pathogenesis and transmissibility of two Asian-origin H5Nx viruses in mammalian animal models. The newly isolated H5N2 and H5N8 viruses were able to cause severe disease in mice only at high doses. Both viruses replicated efficiently in the upper and lower respiratory tracts of ferrets; however clinical symptoms were generally mild and there was no evidence of systemic dissemination of virus to multiple organs. Moreover, these influenza H5Nx viruses lacked the ability to transmit between ferrets in a direct contact setting. We further assessed viral replication kinetics of the novel H5Nx viruses in a human bronchial epithelium cell line, Calu-3. Both H5Nx viruses replicated to a level comparable to a human seasonal H1N1 virus, but significantly lower than a virulent Asian-lineage H5N1 HPAI virus. Although the recently isolated H5N2 and H5N8 viruses displayed moderate pathogenicity in mammalian models, their ability to rapidly spread among avian species, reassort, and generate novel strains underscores the need for continued risk assessment in mammals.

    IMPORTANCE In 2015, highly pathogenic avian influenza (HPAI) H5 viruses have caused outbreaks in domestic poultry in multiple U.S. states. The economic losses incurred with H5N8 and H5N2 subtype virus infection have raised serious concerns for the poultry industry and the general public due to the potential risk of human infection. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals, which is an essential component of pandemic risk assessment. This study demonstrates that the newly isolated H5N2 and H5N8 viruses lacked the ability to transmit between ferrets and exhibited low to moderate virulence in mammals. In human bronchial epithelial (Calu-3) cells, both H5N8 and H5N2 viruses replicated to a level comparable to a human seasonal virus, but significantly lower than a virulent Asian-lineage H5N1 (A/Thailand/16/2004) virus. The results of this study are important for the evaluation of public health risk.

  • Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion F Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine [PublishAheadOfPrint]

  • Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously-described attenuating mutation that was stabilized against de-attenuation: specifically, a non-temperature-sensitivity deletion mutation involving six nucleotides in the overlapping P/C ORFs (C170), or a temperature-sensitivity missense mutation in the L ORF (LY942A). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as wt HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-C170-F1, -F2, and -F3, and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-C170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates and highly restricted in lungs, and were genetically stable in vivo. Among the C170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of over-attenuation. The C170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine.

    IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIVs. We are developing live attenuated RSV and HPIV vaccines for use in virus-naiiuml;ve infants. Live attenuated RSV strains in particular are difficult to develop due to its poor growth and physical instability, but these obstacles could be avoided by the use of a vaccine vector. We describe the development and pre-clinical evaluation of live attenuated rHPIV1 vectors expressing the RSV F protein. Two different attenuated rHPIV1 backbones were engineered to express RSV F from three different gene positions each. The rHPIV1-C170-F1 vector, bearing an attenuating deletion mutation (C170) in the P/C gene and expressing RSV F from the pre-N position, was attenuated, stable, and immunogenic in the hamster model against RSV F protein and HPIV1 and provided substantial protection against RSV challenge. This study provided a candidate rHPIV1-RSV-F vaccine virus suitable for continued development as a bivalent vaccine against two major childhood pathogens.

  • Vaccinia B1 kinase is required for post-replicative stages of the viral lifecycle in a BAF-independent manner in U2OS cells [PublishAheadOfPrint]

  • The vaccinia B1R gene encodes a highly conserved protein kinase that is essential for the poxviral lifecycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector BAF (barrier to autointegration factor or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature sensitive mutant virus (Cts2) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells Cts2 viral DNA replication is unimpaired at non-permissive temperature. However, Cts2 viral yield in these cells is reduced more than 10 fold, thus indicating that B1 is required at another stage of the vaccinia viral lifecycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells, but can be recovered either through overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 infection demonstrates that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncover a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell-type specific regulation of BAF and reveal a role for B1 beyond DNA replication during the late stages of the viral life cycle.

    IMPORTANCE The most well characterized role for the vaccinia B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral lifecycle have been postulated for decades, but are difficult to examine directly due to the importance of B1 during DNA replication. Herein, we demonstrate that in U2OS cells, a B1-mutant virus escapes the block in DNA replication observed in other cell types and instead this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral lifecycle, both dependent and independent of BAF.

  • Elite control, gut CD4 T cell sparing, and enhanced mucosal T cell responses in Macaca nemestrina infected by an SIV lacking a gp41 trafficking motif [PublishAheadOfPrint]

  • Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxOOslash; trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed GY, leads to a striking perturbation in pathogenesis in rhesus macaques (M. mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4+ T cells and an absence of microbial translocation. Here we evaluated GY in pig-tailed macaques (M. nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques GY also replicated acutely to high peak plasma RNA levels identical to SIVmac239, and caused only transient infection of CD4+ T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled GY replication with plasma viral loads of llt;15-50 RNA copies/ml. CD4+ T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust, anti-viral CD4+ T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8+ cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by GY's ablation of the GYxxOOslash; trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of SIV infection.

    IMPORTANCE Pathogenesis of human (HIV) and simian (SIV) immunodeficiency viruses reflects a balance between viral replication host innate and adaptive anti-viral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

  • Adenovirus E4-ORF3 targets PIAS3 and together with E1B-55K remodels SUMO interactions in the nucleus and at virus genome replication domains [PublishAheadOfPrint]

  • Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through non-covalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue E1B-55K/E4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3 associated E2A domains is strikingly different to that in wild type Ad5 infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3 associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, 2, or 4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionary conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication.

    IMPORTANCE SUMO is a key post-translational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection.

  • An Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21Cip1. In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97, and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21Cip1. Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibiting the kinase resulted in elevated levels of p21Cip1 in wild-type but not a pUL27-deficient virus. We manipulated p21Cip1 levels to evaluate the functional consequence to MBV. Overexpression of p21Cip1 restored MBV activity against a pUL27-deficient virus while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21Cip1 in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21Cip1 and support the idea that CDKs can complement some activities of pUL97.

    IMPORTANCE HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist yet exhibit high levels of toxicity, are not approved for use during pregnancy and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21Cip1. In contrast, we provide evidence that p21Cip1 functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21Cip1 activators might be useful in combination with MBV to effectively inhibit HCMV infections.

  • A14U Substitution in 3' Non-Coding Region of M vRNA Supports Replication of NS1-Deleted Influenza Virus by Modulating Alternative Splicing of M mRNAs [PublishAheadOfPrint]

  • The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. NS1-deleted (DelNS1) influenza viruses are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3rrsquo; non-coding region of the M vRNA segment which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus found that lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3 and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts, and provide strategic information important to construction of NS1-deleted vaccine strains.

    IMPORTANCE The non-structural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and NS1 deleted live attenuated viruses show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication competent DelNS1 virus which carries an A14U substitution in the 3rrsquo; non-coding region of the M vRNA segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent NS1-deleted strains for use in functional and vaccine studies.

  • A NOVEL PDZ DOMAIN INTERACTION MEDIATES THE BINDING BETWEEN HPV-16 L2 AND SORTING NEXIN 27 AND MODULATES VIRION TRAFFICKING. [PublishAheadOfPrint]

  • Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-Ferm family member, sorting nexin 27, which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct, and unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that siRNA-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection, but double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.

    IMPORTANCE The trafficking of papillomaviruses to the host cell nucleus during their natural infectious life-cycle is a process that is incompletely understood. Studies have suggested that the virus L2 minor capsid protein can interact with the endosomal recycling pathway, in part, by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-golgi network rather than back to the plasma membrane. In this study we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics as well as providing potential tools to further dissect endosomal structure and function.

  • Reduced SIV replication in macrophages of sooty mangabeys is associated with increased expression of host restriction factors. [PublishAheadOfPrint]

  • Macrophages are target cells of HIV/SIV infection that may play a role in AIDS pathogenesis and contribute to the long-lived reservoir of latently infected cells during antiretroviral therapy (ART). In previous work, we and others have shown that during pathogenic SIV infection of rhesus macaques (RMs), rapid disease progression is associated with high levels of in vivo macrophage infection. In contrast, during nonpathogenic SIV infection of sooty mangabeys (SMs), neither spontaneous nor experimental CD4+ T cell depletion results in substantial levels of in vivo macrophage infection. To test the hypothesis that SM macrophages are intrinsically more resistant to SIV infection than RM macrophages, we undertook an in vitro comparative assessment of monocyte-derived macrophages (MDMs) from both nonhuman primate species. Using the primary isolate SIVM949, which replicates well in lymphocytes from both RMs and SMs, we found that infection of RM macrophages resulted in persistent SIV-RNA production while SIV-RNA levels in SM macrophage cultures decreased 10- to 100-fold over a similar temporal course of in vitro infection. To explore potential mechanisms responsible for the lower levels of SIV replication and/or production in macrophages from SMs we comparatively assessed, in the two studied species, the expression of the SIV co-receptor as well as the expression of a number of host restriction factors. While previous studies showed that SM monocytes express lower levels of CCR5 (but not CD4) than RM monocytes, the level of CCR5 expression in MDMs was similar in the two species. Interestingly, we found that SM macrophages exhibited a significantly greater increase in the expression of tetherin (P=0.003) and TRIM22 (P=0.0006) in response to interferon (IFN)-aalpha; stimulation and increased expression of multiple host restriction factors in response to lipopolysaccharide (LPS) stimulation and exposure to SIV. Overall these findings confirm, in an in vitro infection system, that SM macrophages are relatively more resistant to SIV infection compared to RM macrophages, and suggest that a combination of entry and post-entry restriction mechanisms may protect these cells from productive SIV infection.

    IMPORTANCE This manuscript represents the first in vivo comparative analysis of monocyte-derived macrophages (MDMs) between rhesus macaques, i.e., experimental SIV hosts in which the infection is pathogenic and macrophages can be infected, and sooty mangabeys, i.e., natural SIV hosts in which the infection is non-pathogenic and macrophages are virtually never infected in vivo. This study demonstrates that mangabey-derived MDMs are more resistant to SIV infection in vitro as compared to macaque-derived MDMs, and provides a potential explanation for this observation by showing increased expression of specific retrovirus restriction factors in mangabey-derived macrophages. Overall, this study is important as it contributes to our understanding of why SIV infection is non-pathogenic in sooty mangabeys while it is pathogenic in macaques, and is consistent with a pathogenic role for in vivo macrophage infection during pathogenic lentiviral infection.

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