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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
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  • Current Issue of Journal of Virology

    JVI Current Issue

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  • The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack [Cellular Response to Infection]

  • Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV.

    IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.

  • Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells [Virus-Cell Interactions]

  • Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales. AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti. Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro. The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.

    IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

  • Transmembrane Domains Mediate Intra- and Extracellular Trafficking of Epstein-Barr Virus Latent Membrane Protein 1 [Virus-Cell Interactions]

  • EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.

    IMPORTANCE EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.

  • Expression of a Structural Protein of the Mycovirus FgV-ch9 Negatively Affects the Transcript Level of a Novel Symptom Alleviation Factor and Causes Virus Infection-Like Symptoms in Fusarium graminearum [Virus-Cell Interactions]

  • Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1. This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence.

    IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

  • Human Cytomegalovirus Immediate Early 1 Protein Causes Loss of SOX2 from Neural Progenitor Cells by Trapping Unphosphorylated STAT3 in the Nucleus [Virus-Cell Interactions]

  • The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV infection to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here, we show that HCMV infection of NPCs results in loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 downregulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.

    IMPORTANCE Human cytomegalovirus (HCMV) infections are a leading cause of brain damage, hearing loss, and other neurological disabilities in children. We report that the HCMV proteins known as IE1 and IE2 target expression of human SOX2, a central pluripotency-associated transcription factor that governs neural progenitor cell (NPC) fate and is required for normal brain development. Both during HCMV infection and when expressed alone, IE1 causes the loss of SOX2 from NPCs. IE1 mediates SOX2 depletion by targeting STAT3, a critical upstream regulator of SOX2 expression. Our findings reveal an unanticipated mechanism by which a common virus may cause damage to the developing nervous system and suggest novel targets for medical intervention.

  • Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43 [Virus-Cell Interactions]

  • Human coronaviruses (HCoVs) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under still unclear circumstances. HCoV-induced neuropathologies in humans are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb and piriform cortex and then the brain stem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism is reminiscent of viral assembly sites. We further reveal that HCoV OC43 modes of propagation can be modulated by selected HCoV OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.

    IMPORTANCE Coronaviruses may invade the CNS, disseminate, and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains have been proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV OC43 to access and spread to and within the CNS toward the brain stem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that axonal transport governs HCoV OC43 egress in the CNS, leading to the exacerbation of neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS, as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential druggable molecular virus-host interfaces.

  • Transgene-Assisted Genetic Screen Identifies rsd-6 and Novel Genes as Key Components of Antiviral RNA Interference in Caenorhabditis elegans [Pathogenesis and Immunity]

  • RNA interference (RNAi) is a widespread antiviral mechanism triggered by virus-produced double-stranded RNAs (dsRNAs). In Caenorhabditis elegans, antiviral RNAi involves a RIG-I-like RNA helicase, termed DRH-1 (dicer related RNA helicase 1), that is not required for classical RNAi triggered by artificial dsRNA. Currently, whether antiviral RNAi in C. elegans involves novel factors that are dispensable for classical RNAi remains an open question. To address this question, we designed and carried out a genetic screen that aims to identify novel genes involved in worm antiviral RNAi. By introducing extra copies of known antiviral RNAi genes into the reporter worms, we managed to reject alleles derived from 4 known antiviral RNAi genes, including the DRH-1 coding gene, during the screen. Our genetic screen altogether identified 25 alleles, which were assigned to 11 candidate genes and 2 known antiviral RNAi genes through genetic complementation tests. Using a mapping-by-sequencing strategy, we identified one of the candidate genes as rsd-6, a gene that helps maintain genome integrity through an endogenous gene-silencing pathway but was not known to be required for antiviral RNAi. More importantly, we found that two of the candidate genes are required for antiviral RNAi targeting Orsay virus, a natural viral pathogen of C. elegans, but dispensable for classical RNAi. Since drh-1 is so far the only antiviral RNAi gene not required for classical RNAi, we believe that our genetic screen led to identification of novel worm genes that may target virus-specific features to function in RNAi.

    IMPORTANCE In nematode worms, drh-1 detects virus-produced double-stranded RNA (dsRNA), thereby specifically contributing to antiviral RNA silencing. To identify drh-1-like genes with dedicated function in antiviral RNAi, we recently carried out a genetic screen that was designed to automatically reject all alleles derived from 4 known antiviral silencing genes, including drh-1. Of the 11 candidate genes identified, we found two of them to be required for antiviral silencing targeting a natural viral pathogen of C. elegans but not for classical RNA silencing triggered by artificial dsRNA. We believe that these two genes are novel components of worm antiviral RNAi, considering the fact that drh-1 is the only known antiviral RNAi gene that is dispensable for classical RNAi. This genetic screen also identified rsd-6, a gene that maintains genome integrity under unfavorable conditions, as a key regulator of worm antiviral silencing, demonstrating an interplay between antiviral immunity and genome integrity maintenance.

  • Role of MxB in Alpha Interferon-Mediated Inhibition of HIV-1 Infection [Virus-Cell Interactions]

  • Type I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins.

    IMPORTANCE The results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4+ T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors.

  • A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks [Genetic Diversity and Evolution]

  • Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses, such as Japanese encephalitis virus, West Nile virus, and Bagaza virus, is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks, while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks.

    IMPORTANCE Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

  • Long Noncoding RNA ITPRIP-1 Positively Regulates the Innate Immune Response through Promotion of Oligomerization and Activation of MDA5 [Pathogenesis and Immunity]

  • Emerging evidence indicates that long noncoding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and the interferon (IFN) pathway remains largely unknown. Here, we report that lncRNA ITPRIP-1 (lncITPRIP-1) is involved in viral infection and plays a crucial role in the virus-triggered IFN signaling pathway through the targeting of melanoma differentiation-associated gene 5 (MDA5). LncITPRIP-1 can be induced by viral infection, which is not entirely dependent on the IFN signal. Besides, there is no coding potential found in the lncITPRIP-1 transcript. LncITPRIP-1 binds to the C terminus of MDA5, and it possesses the ability to boost the oligomerization of both the full length and the 2 caspase activation and recruitment domains of MDA5 in a K63-linked polyubiquitination-independent manner. Amazingly, we also found that MDA5 can suppress hepatitis C virus (HCV) replication independently of IFN signaling through its C-terminal-deficient domain bound to viral RNA, in which lncITPRIP-1 plays a role as an assistant. In addition, the expression of lncITPRIP-1 is highly consistent with MDA5 expression, indicating that lncITPRIP-1 may function as a cofactor of MDA5. All the data suggest that lncITPRIP-1 enhances the innate immune response to viral infection through the promotion of oligomerization and activation of MDA5. Our study discovers the first lncRNA ITPRIP-1 involved in MDA5 activation.

    IMPORTANCE Hepatitis C virus infection is a global health issue, and there is still no available vaccine, which makes it urgent to reveal the underlying mechanisms of HCV and host factors. Although RIG-I has been recognized as the leading cytoplasmic sensor against HCV for a long time, recent findings that MDA5 regulates the IFN response to HCV have emerged. Our work validates the significant role of MDA5 in IFN signaling and HCV infection and proposes the first lncRNA inhibiting HCV replication by promoting the activation of MDA5 and mediating the association between MDA5 and HCV RNA, the study of which may shed light on the MDA5 function and treatment for hepatitis C patients. Our suggested model of how lncITPRIP-1 orchestrates signal transduction for IFN production illustrates the essential role of lncRNAs in virus elimination.

  • Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions [Virus-Cell Interactions]

  • Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity.

    IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.

  • Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9 [Virus-Cell Interactions]

  • Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.

    IMPORTANCE In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.

  • Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression [Gene Delivery]

  • Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.

    IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.

  • Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2{alpha} To Activate Productive Viral Replication [Virus-Cell Interactions]

  • Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) causes the angiogenic tumor KS and two B-cell malignancies. The KSHV nonstructural membrane protein encoded by the open reading frame (ORF) K15 recruits and activates several cellular proteins, including phospholipase C1 (PLC1), components of the NF-B pathway, as well as members of the Src family of nonreceptor tyrosine kinases, and thereby plays an important role in the activation of angiogenic and inflammatory pathways that contribute to the pathogenesis of KS as well as KSHV productive (lytic) replication. In order to identify novel cellular components involved in the biology of pK15, we immunoprecipitated pK15 from KSHV-infected endothelial cells and identified associated proteins by label-free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes, such as the endocytic pathway, that could be involved in the function of pK15. We found that the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2aalpha;, which is involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, interacts and colocalizes with pK15 in vesicular structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2aalpha; contributes to the pK15-dependent phosphorylation of PLC1 and Erk1/2. PI3K-C2aalpha; also plays a role in KSHV lytic replication, as evidenced by the reduced expression of the viral lytic genes K-bZIP and ORF45 as well as the reduced release of infectious virus in PI3K-C2aalpha;-depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2aalpha; protein in the functional properties of the KSHV pK15 protein.

    IMPORTANCE The nonstructural membrane protein encoded by open reading frame K15 of Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV8) activates several intracellular signaling pathways that contribute to the angiogenic properties of KSHV in endothelial cells and to its reactivation from latency. A detailed understanding of how pK15 activates these intracellular signaling pathways is a prerequisite for targeting these processes specifically in KSHV-infected cells. By identifying pK15-associated cellular proteins using a combination of immunoprecipitation and mass spectrometry, we provide evidence that pK15-dependent signaling may occur from intracellular vesicles and rely on the endocytotic machinery. Specifically, a class II PI3K, PI3K-C2aalpha;, is recruited by pK15 and involved in pK15-dependent intracellular signaling and viral reactivation from latency. These findings are of importance for future intervention strategies that aim to disrupt the activation of intracellular signaling by pK15 in order to antagonize KSHV productive replication and tumorigenesis.

  • Structural Basis for the Broad, Antibody-Mediated Neutralization of H5N1 Influenza Virus [Structure and Assembly]

  • Human infection with highly pathogenic avian influenza A viruses causes severe disease and fatalities. We previously identified a potent and broadly neutralizing antibody (bnAb), 13D4, against the H5N1 virus. Here, we report the co-crystal structure of 13D4 in complex with the hemagglutinin (HA) of A/Vietnam/1194/2004 (H5N1). We show that heavy-chain complementarity-determining region 3 (HCDR3) of 13D4 confers broad yet specific neutralization against H5N1, undergoing conformational rearrangement to bind to the receptor binding site (RBS). Further, we show that mutating four critical residues within the RBSmmdash;Trp153, Lys156, Lys193, and Leu194mmdash;disrupts the binding between 13D4 and HA. Viruses bearing Asn193 instead of Lys/Arg can evade 13D4 neutralization, indicating that Lys193 polymorphism might be, at least in part, involved in the antigenicity of recent H5 genotypes (such as H5N6 and H5N8) as distinguished from H5N1. BnAb 13D4 may offers a template for therapeutic RBS inhibitor design and serve as an indicator of antigenic change for current H5 viruses.

    IMPORTANCE Infection by highly pathogenic avian influenza A virus remains a threat to public health. Our broadly neutralizing antibody, 13D4, is capable of neutralizing all representative H5N1 viruses and protecting mice against lethal challenge. Structural analysis revealed that 13D4 uses heavy-chain complementarity-determining region 3 (HCDR3) to fit the receptor binding site (RBS) via conformational rearrangement. Four conserved residues within the RBS are critical for the broad potency of 13D4. Importantly, polymorphism of Lys193 on the RBS may be associated with the antigenicity shift from H5N1 to other newly emerging viruses, such as H5N6 and H5N8. Our findings may pave the way for highly pathogenic avian influenza virus vaccine development and therapeutic RBS inhibitor design.

  • Marginal Effects of Systemic CCR5 Blockade with Maraviroc on Oral Simian Immunodeficiency Virus Transmission to Infant Macaques [Pathogenesis and Immunity]

  • Current approaches do not eliminate all human immunodeficiency virus type 1 (HIV-1) maternal-to-infant transmissions (MTIT); new prevention paradigms might help avert new infections. We administered maraviroc (MVC) to rhesus macaques (RMs) to block CCR5-mediated entry, followed by repeated oral exposure of a CCR5-dependent clone of simian immunodeficiency virus (SIV) mac251 (SIVmac766). MVC significantly blocked the CCR5 coreceptor in peripheral blood mononuclear cells and tissue cells. All control animals and 60% of MVC-treated infant RMs became infected by the 6th challenge, with no significant difference between the number of exposures (P = 0.15). At the time of viral exposures, MVC plasma and tissue (including tonsil) concentrations were within the range seen in humans receiving MVC as a therapeutic. Both treated and control RMs were infected with only a single transmitted/founder variant, consistent with the dose of virus typical of HIV-1 infection. The uninfected RMs expressed the lowest levels of CCR5 on the CD4+ T cells. Ramp-up viremia was significantly delayed (P = 0.05) in the MVC-treated RMs, yet peak and postpeak viral loads were similar in treated and control RMs. In conclusion, in spite of apparent effective CCR5 blockade in infant RMs, MVC had a marginal impact on acquisition and only a minimal impact on the postinfection delay of viremia following oral SIV infection. Newly developed, more effective CCR5 blockers may have a more dramatic impact on oral SIV transmission than MVC.

    IMPORTANCE We have previously suggested that the very low levels of simian immunodeficiency virus (SIV) maternal-to-infant transmissions (MTIT) in African nonhuman primates that are natural hosts of SIVs are due to a low availability of target cells (CCR5+ CD4+ T cells) in the oral mucosa of the infants, rather than maternal and milk factors. To confirm this new MTIT paradigm, we performed a proof-of-concept study in which we therapeutically blocked CCR5 with maraviroc (MVC) and orally exposed MVC-treated and naive infant rhesus macaques to SIV. MVC had only a marginal effect on oral SIV transmission. However, the observation that the infant RMs that remained uninfected at the completion of the study, after 6 repeated viral challenges, had the lowest CCR5 expression on the CD4+ T cells prior to the MVC treatment appears to confirm our hypothesis, also suggesting that the partial effect of MVC is due to a limited efficacy of the drug. New, more effective CCR5 inhibitors may have a better effect in preventing SIV and HIV transmission.

  • Transcriptional and Translational Landscape of Equine Torovirus [Genome Replication and Regulation of Viral Gene Expression]

  • The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5' untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.

    IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames "hidden" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

  • Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity [Structure and Assembly]

  • Coronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-N7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-N7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.

    IMPORTANCE Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.

  • Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models [Vaccines and Antiviral Agents]

  • Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ggt;ggt; Min E ggt; Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.

    IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barreeacute; syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

  • Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines [Vaccines and Antiviral Agents]

  • With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2'O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2'O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2'O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines.

    IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2'O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2'O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2'O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

  • Murine Pneumonia Virus Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus from an Added Gene Is Highly Attenuated and Immunogenic in Rhesus Macaques [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae. Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.

    IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro. In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.

  • Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors Has a High Genetic Barrier and Severe Fitness Costs [Virus-Cell Interactions]

  • VIRIP has been identified as natural HIV-1 inhibitor targeting the gp41 fusion peptide. An optimized analogue (VIR-576) was effective in a phase I/II clinical trial and initial studies showed that HIV-1 resistance to VIRIP-based inhibitors has a high genetic barrier. Partially resistant CXCR4 (X4)-tropic HIV-1 NL4-3 variants could be obtained, however, after more than 15 months of passaging in MT-4 cells in the presence of another derivative (VIR-353). Sequence analyses identified the accumulation of seven mutations across the HIV-1 envelope glycoprotein but outside the gp41 fusion peptide. The authors suggested that the three initial alterations conferred resistance, while subsequent changes restored viral fitness. Here, we introduced these mutations individually and in combination into X4- and CCR5 (R5)-tropic HIV-1 constructs and determined their impact on VIR-353 and VIR-576 susceptibility, viral infectivity, replication fitness, and fusogenicity. We found that essentially all seven mutations contribute to reduced susceptibility to VIRIP-based inhibitors. HIV-1 constructs containing gge;4 changes were substantially more resistant to both VIRIP-based inhibitors and the VRC34.01 antibody targeting the fusion peptide. However, they were also much less infectious and fusogenic than those harboring only the three initial alterations. Furthermore, the additional changes attenuated rather than rescued HIV-1 replication in primary human cells. Thus, the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is higher than previously suggested, and mutations reducing viral susceptibility come at a severe fitness cost that was not rescued during long-term cell culture passage.

    IMPORTANCE Many viral pathogens are critically dependent on fusion peptides (FPs) that are inserted into the cellular membrane for infection. Initially, it was thought that FPs cannot be targeted for therapy because they are hardly accessible. However, an optimized derivative (VIR-576) of an endogenous fragment of aalpha;1-antitrypsin, named VIRIP, targeting the gp41 FP reduced viral loads in HIV-1-infected individuals. Characterization of HIV-1 variants selected during long-term cell-culture passage in the presence of a VIRIP derivative suggested that just three mutations in the HIV-1 Env protein might be sufficient for VIRIP resistance and that four subsequent changes restored viral fitness. Here, we show that all seven mutations contribute to reduced viral susceptibility to VIRIP-based inhibitors and demonstrate that the additional changes strongly impair rather than rescue HIV-1 infectivity, fusogenicity, and replication fitness. High genetic barrier to resistance and severe fitness cost support further clinical development of this class of antiviral agents.

  • Dissecting the Herpesvirus Architecture by Targeted Proteolysis [Structure and Assembly]

  • Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of various amounts of 20 or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein, a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized, and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36, whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.

    IMPORTANCE Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument, and with it we begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

  • Viral Discovery in the Invasive Australian Cane Toad (Rhinella marina) Using Metatranscriptomic and Genomic Approaches [Genetic Diversity and Evolution]

  • Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. The release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile- and amphibian-specific cluster of the Picornaviridae basal to the Kobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, Rhinella marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains 8 complete RMERV proviruses as well as 21 additional truncated insertions. The oldest full-length RMERV provirus was estimated to have inserted 1.9 million years ago (MYA). To screen for these viral sequences in additional toads, we analyzed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, while RMERV transcripts were observed at all six sites. Finally, we scanned the cane toad genome for nonretroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae. This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.

    IMPORTANCE Cane toads are poisonous amphibians that were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threaten many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality rates, yet few cane toad viruses have been characterized. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences that were genetically similar to viruses infecting frogs, reptiles, and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies that investigate their prevalence and potential as agents for biocontrol.

  • A Dual Motif in the Hemagglutinin of H5N1 Goose/Guangdong-Like Highly Pathogenic Avian Influenza Virus Strains Is Conserved from Their Early Evolution and Increases both Membrane Fusion pH and Virulence [Pathogenesis and Immunity]

  • Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by the acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required coadaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3,385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R and 124I but a frequency of 97.5% for the dual combination. This highly conserved dual motif is still retained in contemporary H5 HPAIV, including the novel H5NX reassortants carrying neuraminidases of different subtypes, like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/1997, carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S and124T, characteristic of low-pathogenic strains, prevented virus rescue, while the single substitutions attenuated the virus in both chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less-acidic endosomes, e.g., within the respiratory tract, resulting in an extended organ tropism. Taken together, HA coadaptation to increased acid sensitivity promoted the early evolution of H5 Goose/Guangdong-like HPAIV strains and is still required for their zoonotic potential.

    IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required coadaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic of low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less-acidic endosomes, e.g., within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted the emergence of H5 Goose/Guangdong-like HPAIV strains and is required for their zoonotic potential.

  • A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins {alpha}3{beta}1, {alpha}V{beta}3, and {alpha}V{beta}5 [Virus-Cell Interactions]

  • Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins aalpha;3bbeta;1, aalpha;Vbbeta;3, and aalpha;Vbbeta;5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of aalpha;V- and bbeta;1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.

    IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

  • Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis [Pathogenesis and Immunity]

  • UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus 1 (HSV-1) and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been reported, there is a lack of information on how these viral protein kinases are regulated in HSV-infected cells. In this study, we used a large-scale phosphoproteomic analysis of HSV-2-infected cells to identify a physiological phosphorylation site in HSV-2 UL13 (i.e., Ser-18) and investigated the significance of phosphorylation of this site in HSV-2-infected cell cultures and mice. Our results were as follows. (i) An alanine substitution at UL13 Ser-18 (S18A) significantly reduced HSV-2 replication and cell-to-cell spread in U2OS cells to a level similar to those of the UL13-null and kinase-dead mutations. (ii) The UL13 S18A mutation significantly impaired phosphorylation of a cellular substrate of this viral protein kinase in HSV-2-infected U2OS cells. (iii) Following vaginal infection of mice, the UL13 S18A mutation significantly reduced mortality, HSV-2 replication in the vagina, and development of vaginal disease to levels similar to those of the UL13-null and the kinase-dead mutations. (iv) A phosphomimetic substitution at UL13 Ser-18 significantly restored the phenotype observed with the UL13 S18A mutation in U2OS cells and mice. Collectively, our results suggested that phosphorylation of UL13 Ser-18 regulated UL13 function in HSV-2-infected cells and that this regulation was critical for the functional activity of HSV-2 UL13 in vitro and in vivo and also for HSV-2 replication and pathogenesis.

    IMPORTANCE Based on studies on cellular protein kinases, it is obvious that the regulatory mechanisms of protein kinases are as crucial as their functional consequences. Herpesviruses each encode at least one protein kinase, but the mechanism by which these kinases are regulated in infected cells remains to be elucidated, with a few exceptions, although information on their functional effects has been accumulating. In this study, we have shown that phosphorylation of the HSV-2 UL13 protein kinase at Ser-18 regulated its function in infected cells, and this regulation was critical for HSV-2 replication and pathogenesis in vivo. UL13 is conserved in all members of the family Herpesviridae, and this is the first report clarifying the regulatory mechanism of a conserved herpesvirus protein kinase that is involved in viral replication and pathogenesis in vivo. Our study may provide insight into the regulatory mechanisms of the other conserved herpesvirus protein kinases.

  • Multiple Posttranscriptional Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease [Virus-Cell Interactions]

  • The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with the aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from cotransfected plasmids were also retained in the same nuclei where vhs mRNA was located, while poly(A) binding protein (PABP) was relocalized to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Coexpression of VP16 and VP22 rescued the cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous green fluorescent protein transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and autoinduced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.

    IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV-1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs but that must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted posttranscriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and coexpressed mRNAs for nuclear retention, an activity that is relieved by coexpression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors coordinate gene expression at the time that they are needed. These findings are broadly relevant to both virus and cellular gene expression.

  • Correction for Chung et al., "Downregulation of Poly(ADP-Ribose) Polymerase 1 by a Viral Processivity Factor Facilitates Lytic Replication of Gammaherpesvirus" [Author Correction]

  • APOBEC3G-Regulated Host Factors Interfere with Measles Virus Replication: Role of REDD1 and Mammalian TORC1 Inhibition [Virus-Cell Interactions]

  • We found earlier that ectopic expression of the cytidine deaminase APOBEC3G (A3G) in Vero cells inhibits measles virus (MV), respiratory syncytial virus, and mumps virus, while the mechanism of inhibition remained unclear. A microarray analysis revealed that in A3G-transduced Vero cells, several cellular transcripts were differentially expressed, suggesting that A3G regulates the expression of host factors. One of the most upregulated host cell factors, REDD1 (regulated in development and DNA damage response-1, also called DDIT4), reduced MV replication ~10-fold upon overexpression in Vero cells. REDD1 is an endogenous inhibitor of mTORC1 (mammalian target of rapamycin complex-1), the central regulator of cellular metabolism. Interestingly, rapamycin reduced the MV replication similarly to REDD1 overexpression, while the combination of both did not lead to further inhibition, suggesting that the same pathway is affected. REDD1 silencing in A3G-expressing Vero cells abolished the inhibitory effect of A3G. In addition, silencing of A3G led to reduced REDD1 expression, confirming that its expression is regulated by A3G. In primary human peripheral blood lymphocytes (PBL), expression of A3G and REDD1 was found to be stimulated by phytohemagglutinin (PHA) and interleukin-2. Small interfering RNA (siRNA)-mediated depletion of A3G in PHA-stimulated PBL reduced REDD1 expression and increased viral titers, which corroborates our findings in Vero cells. Silencing of REDD1 also increased viral titers, confirming the antiviral role of REDD1. Finally, pharmacological inhibition of mTORC1 by rapamycin in PHA-stimulated PBL reduced viral replication to the level found in unstimulated lymphocytes, indicating that mTORC1 activity supports MV replication as a proviral host factor.

    IMPORTANCE Knowledge about host factors supporting or restricting virus replication is required for a deeper understanding of virus-cell interactions and may eventually provide the basis for therapeutic intervention. This work was undertaken predominantly to explain the mechanism of A3G-mediated inhibition of MV, a negative-strand RNA virus that is not affected by the deaminase activity of A3G acting on single-stranded DNA. We found that A3G regulates the expression of several cellular proteins, which influences the capacity of the host cell to replicate MV. One of these, REDD1, which modulates the cellular metabolism in a central position by regulating the kinase complex mTORC1, was identified as the major cellular factor impairing MV replication. These findings show interesting aspects of the function of A3G and the dependence of the MV replication on the metabolic state of the cell. Interestingly, pharmacological inhibition of mTORC1 can be utilized to inhibit MV replication in Vero cells and primary human peripheral blood lymphocytes.

  • HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes [Pathogenesis and Immunity]

  • We have previously shown that HIV-1-infected children develop broader and more potent neutralizing antibody responses than adults. This study aimed to determine the antibody specificities in 16 HIV-1 subtype C-infected children who displayed exceptional neutralization breadth on a 22-multisubtype virus panel. All children were antiretroviral treatment (ART) naive with normal CD4 counts despite being infected for a median of 10.1 years with high viral loads. The specificity of broadly neutralizing antibodies (bNAbs) was determined using epitope-ablating mutants, chimeric constructs, and depletion or inhibition of activity with peptides and glycoproteins. We found that bNAbs in children largely targeted previously defined epitopes, including the V2-glycan, V3-glycan, CD4bs, and gp120-gp41 interface. Remarkably, 63% of children had antibodies targeting 2 or 3 and, in one case, 4 of these bNAb epitopes. Longitudinal analysis of plasma from a mother-child pair over 9 years showed that while they both had similar neutralization profiles, the antibody specificities differed. The mother developed antibodies targeting the V2-glycan and CD4bs, whereas bNAb specificities in the child could not be mapped until 6 years, when a minor V2-glycan response appeared. The child also developed high-titer membrane-proximal external region (MPER) binding antibodies not seen in the mother, although these were not a major bNAb specificity. Overall, exceptional neutralization breadth in this group of children may be the result of extended exposure to high antigenic load in the context of an intact immune system, which allowed for the activation of multiple B cell lineages and the generation of polyclonal responses targeting several bNAb epitopes.

    IMPORTANCE An HIV vaccine is likely to require bNAbs, which have been shown to prevent HIV acquisition in nonhuman primates. Recent evidence suggests that HIV-infected children are inherently better at generating bNAbs than adults. Here, we show that exceptional neutralization breadth in a group of viremic HIV-1 subtype C-infected children was due to the presence of polyclonal bNAb responses. These bNAbs targeted multiple epitopes on the HIV envelope glycoprotein previously defined in adult infection, suggesting that the immature immune system recognizes HIV antigens similarly. Since elicitation of a polyclonal bNAb response is the basis of next-generation HIV envelope vaccines, further studies of how bNAb lineages are stimulated in children is warranted. Furthermore, our findings suggest that children may respond particularly well to vaccines designed to elicit antibodies to multiple bNAb epitopes.

  • Correction for Parolini et al., "Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity" [Author Correction]

  • Rubella Viruses Shift Cellular Bioenergetics to a More Oxidative and Glycolytic Phenotype with a Strain-Specific Requirement for Glutamine [Virus-Cell Interactions]

  • The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on the oxygen consumption rate) and glycolytic (based on the extracellular acidification rate) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of RVs to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population.

    IMPORTANCE RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data add viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. In addition, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations.

  • Recurrent Loss of APOBEC3H Activity during Primate Evolution [Virus-Cell Interactions]

  • Genes in the APOBEC3 family encode cytidine deaminases that provide a barrier against viral infection and retrotransposition. Of all the APOBEC3 genes in humans, APOBEC3H (A3H) is the most polymorphic: some genes encode stable and active A3H proteins, while others are unstable and poorly antiviral. Such variation in human A3H affects interactions with the lentiviral antagonist Vif, which counteracts A3H via proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions, as well as its evolution in Old World monkeys, we characterized A3H variation within four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of function was partially related to protein expression levels but was also influenced by amino acid mutations in the N terminus. Moreover, we demonstrate that the evolution of A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests that the activity of A3H is evolutionarily dynamic and may have a negative effect on host fitness, resulting in its recurrent loss in primates.

    IMPORTANCE Adaptation of viruses to their hosts is critical for viral transmission between different species. Previous studies had identified changes in a protein from the APOBEC3 family that influenced the species specificity of simian immunodeficiency viruses (SIVs) in African green monkeys. We studied the evolution of a related protein in the same system, APOBEC3H, which has experienced a loss of function in humans. This evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken place during primate evolution, suggesting that APOBEC3H places a fitness cost on hosts. The variability of APOBEC3H activity between different primates highlights the differential selective pressures on the APOBEC3 gene family.

  • Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis [Vaccines and Antiviral Agents]

  • The poliovirus eradication initiative has spawned global immunization infrastructure and dramatically decreased the prevalence of the disease, yet the original virus eradication goal has not been met. The suboptimal properties of the existing vaccines are among the major reasons why the program has repeatedly missed eradication deadlines. Oral live poliovirus vaccine (OPV), while affordable and effective, occasionally causes the disease in the primary recipients, and the attenuated viruses rapidly regain virulence and can cause poliomyelitis outbreaks. Inactivated poliovirus vaccine (IPV) is safe but expensive and does not induce the mucosal immunity necessary to interrupt virus transmission. While the need for a better vaccine is widely recognized, current efforts are focused largely on improvements to the OPV or IPV, which are still beset by the fundamental drawbacks of the original products. Here we demonstrate a different design of an antipoliovirus vaccine based on in situ production of virus-like particles (VLPs). The poliovirus capsid protein precursor, together with a protease required for its processing, are expressed from a Newcastle disease virus (NDV) vector, a negative-strand RNA virus with mucosal tropism. In this system, poliovirus VLPs are produced in the cells of vaccine recipients and are presented to their immune systems in the context of active replication of NDV, which serves as a natural adjuvant. Intranasal administration of the vectored vaccine to guinea pigs induced strong neutralizing systemic and mucosal antibody responses. Thus, the vectored poliovirus vaccine combines the affordability and efficiency of a live vaccine with absolute safety, since no full-length poliovirus genome is present at any stage of the vaccine life cycle.

    IMPORTANCE A new, safe, and effective vaccine against poliovirus is urgently needed not only to complete the eradication of the virus but also to be used in the future to prevent possible virus reemergence in a postpolio world. Currently, new formulations of the oral vaccine, as well as improvements to the inactivated vaccine, are being explored. In this study, we designed a viral vector with mucosal tropism that expresses poliovirus capsid proteins. Thus, poliovirus VLPs are produced in vivo, in the cells of a vaccine recipient, and are presented to the immune system in the context of vector virus replication, stimulating the development of systemic and mucosal immune responses. Such an approach allows the development of an affordable and safe vaccine that does not rely on the full-length poliovirus genome at any stage.

  • The Cellular Coactivator HCF-1 Is Required for Glucocorticoid Receptor-Mediated Transcription of Bovine Herpesvirus 1 Immediate Early Genes [Genome Replication and Regulation of Viral Gene Expression]

  • Following productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo. As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1nndash;GR mechanism in the absence of the viral IE activator VP16.

    IMPORTANCE BoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1nndash;GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.

  • Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype [Vaccines and Antiviral Agents]

  • Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.

    IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

  • The Methyltransferase-Like Domain of Chikungunya Virus nsP2 Inhibits the Interferon Response by Promoting the Nuclear Export of STAT1 [Virus-Cell Interactions]

  • Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has evolved effective mechanisms to counteract the type I interferon (IFN) response. Upon recognition of the virus, cells secrete IFNs, which signal through transmembrane receptors (IFNAR) to phosphorylate STAT proteins (pSTAT). pSTAT dimers are transported into the nucleus by importin-aalpha;5 and activate the transcription of IFN-stimulated genes (ISGs), increasing cellular resistance to infection. Subsequently, STAT proteins are shuttled back into the cytoplasm by the exportin CRM1. CHIKV nonstructural protein 2 (nsP2) reduces ISG expression by inhibiting general host cell transcription and by specifically reducing the levels of nuclear pSTAT1 via an unknown mechanism. To systematically examine where nsP2 acts within the JAK/STAT signaling cascade, we used two well-characterized mutants of nsP2, P718S and KR649AA. Both mutations abrogate nsP2's ability to shut off host transcription, but only the KR649AA mutant localizes exclusively to the cytoplasm and no longer specifically inhibits JAK/STAT signaling. These mutant nsP2 proteins did not differentially affect IFNAR expression levels or STAT1 phosphorylation in response to IFNs. Coimmunoprecipitation experiments showed that in the presence of nsP2, STAT1 still effectively bound importin-aalpha;5. Chemically blocking CRM1-mediated nuclear export in the presence of nsP2 additionally showed that nuclear translocation of STAT1 is not affected by nsP2. nsP2 putatively has five domains. Redirecting the nsP2 KR649AA mutant or just nsP2's C-terminal methyltransferase-like domain into the nucleus strongly reduced nuclear pSTAT in response to IFN stimulation. This demonstrates that the C-terminal domain of nuclear nsP2 specifically inhibits the IFN response by promoting the nuclear export of STAT1.

    IMPORTANCE Chikungunya virus is an emerging pathogen associated with large outbreaks on the African, Asian, European, and both American continents. In most patients, infection results in high fever, rash, and incapacitating (chronic) arthralgia. CHIKV effectively inhibits the first line of defense, the innate immune response. As a result, stimulation of the innate immune response with interferons (IFNs) is ineffective as a treatment for CHIKV disease. The IFN response requires an intact downstream signaling cascade called the JAK/STAT signaling pathway, which is effectively inhibited by CHIKV nonstructural protein 2 (nsP2) via an unknown mechanism. The research described here specifies where in the JAK/STAT signaling cascade the IFN response is inhibited and which protein domain of nsP2 is responsible for IFN inhibition. The results illuminate new aspects of antiviral defense and CHIKV counterdefense strategies and will direct the search for novel antiviral compounds.

  • Prolonged Evolution of Virus-Specific Memory T Cell Immunity after Severe Avian Influenza A (H7N9) Virus Infection [Pathogenesis and Immunity]

  • Since 2013, influenza A H7N9 virus has emerged as the most common avian influenza virus subtype causing human infection, and it is associated with a high fatality risk. However, the characteristics of immune memory in patients who have recovered from H7N9 infection are not well understood. We assembled a cohort of 45 H7N9 survivors followed for up to 15 months after infection. Humoral and cellular immune responses were analyzed in sequential samples obtained at 1.5 to 4 months, 6 to 8 months, and 12 to 15 months postinfection. H7N9-specific antibody concentrations declined over time, and protective antibodies persisted longer in severely ill patients admitted to the intensive care unit (ICU) and patients presenting with acute respiratory distress syndrome (ARDS) than in patients with mild disease. Frequencies of virus-specific gamma interferon (IFN-)-secreting T cells were lower in critically ill patients requiring ventilation than in patients without ventilation within 4 months after infection. The percentages of H7N9-specific IFN--secreting T cells tended to increase over time in patients gge;60 years or in critically ill patients requiring ventilation. Elevated levels of antigen-specific CD8+ T cells expressing the lung-homing marker CD49a were observed at 6 to 8 months after H7N9 infection compared to those in samples obtained at 1.5 to 4 months. Our findings indicate the prolonged reconstruction and evolution of virus-specific T cell immunity in older or critically ill patients and have implications for T cell-directed immunization strategies.

    IMPORTANCE Avian influenza A H7N9 virus remains a major threat to public health. However, no previous studies have determined the characteristics and dynamics of virus-specific T cell immune memory in patients who have recovered from H7N9 infection. Our findings showed that establishment of H7N9-specific T cell memory after H7N9 infection was prolonged in older and severely affected patients. Severely ill patients mounted lower T cell responses in the first 4 months after infection, while T cell responses tended to increase over time in older and severely ill patients. Higher levels of antigen-specific CD8+ T cells expressing the lung-homing marker CD49a were detected at 6 to 8 months after infection. Our results indicated a long-term impact of H7N9 infection on virus-specific memory T cells. These findings advance our understanding of the dynamics of virus-specific memory T cell immunity after H7N9 infection, which is relevant to the development of T cell-based universal influenza vaccines.

  • Acidic pH Mediates Changes in Antigenic and Oligomeric Conformation of Herpes Simplex Virus gB and Is a Determinant of Cell-Specific Entry [Virus-Cell Interactions]

  • Herpes simplex virus (HSV) is an important human pathogen with a high worldwide seroprevalence. HSV enters epithelial cells, the primary site of infection, by a low-pH pathway. HSV glycoprotein B (gB) undergoes low pH-induced conformational changes, which are thought to drive membrane fusion. When neutralized back to physiological pH, these changes become reversible. Here, HSV-infected cells were subjected to short pulses of radiolabeling, followed by immunoprecipitation with a panel of gB monoclonal antibodies (MAbs), demonstrating that gB folds and oligomerizes rapidly and cotranslationally in the endoplasmic reticulum. Full-length gB from transfected cells underwent low-pH-triggered changes in oligomeric conformation in the absence of other viral proteins. MAbs to gB neutralized HSV entry into cells regardless of the pH dependence of the entry pathway, suggesting a conservation of gB function in distinct fusion mechanisms. The combination of heat and acidic pH triggered irreversible changes in the antigenic conformation of the gB fusion domain, while changes in the gB oligomer remained reversible. An elevated temperature alone was not sufficient to induce gB conformational change. Together, these results shed light on the conformation and function of the HSV-1 gB oligomer, which serves as part of the core fusion machinery during viral entry.

    IMPORTANCE Herpes simplex virus (HSV) causes infection of the mouth, skin, eyes, and genitals and establishes lifelong latency in humans. gB is conserved among all herpesviruses. HSV gB undergoes reversible conformational changes following exposure to acidic pH which are thought to mediate fusion and entry into epithelial cells. Here, we identified cotranslational folding and oligomerization of newly synthesized gB. A panel of antibodies to gB blocked both low-pH and pH-neutral entry of HSV, suggesting conserved conformational changes in gB regardless of cell entry route. Changes in HSV gB conformation were not triggered by increased temperature alone, in contrast to results with EBV gB. Acid pH-induced changes in the oligomeric conformation of gB are related but distinct from pH-triggered changes in gB antigenic conformation. These results highlight critical aspects of the class III fusion protein, gB, and inform strategies to block HSV infection at the level of fusion and entry.

  • Attenuated Herpes Simplex Virus 1 (HSV-1) Expressing a Mutant Form of ICP6 Stimulates a Strong Immune Response That Protects Mice against HSV-1-Induced Corneal Disease [Vaccines and Antiviral Agents]

  • We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39, resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0nndash;, ICP8nndash;, and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8nndash; virus, but not the ICP0nndash; virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases.

    IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0nndash;) virus and a replication-incompetent (ICP8nndash;) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development.

  • Human MxB Protein Is a Pan-herpesvirus Restriction Factor [Virus-Cell Interactions]

  • Herpesvirus infections are highly prevalent in the human population and persist for life. They are often acquired subclinically but potentially progress to life-threatening diseases in immunocompromised individuals. The interferon system is indispensable for the control of herpesviral replication. However, the responsible antiviral effector mechanisms are not well characterized. The type I interferon-induced, human myxovirus resistance 2 (MX2) gene product MxB, a dynamin-like large GTPase, has recently been identified as a potent inhibitor of HIV-1. We now show that MxB also interferes with an early step of herpesvirus replication, affecting alpha-, beta-, and gammaherpesviruses before or at the time of immediate early gene expression. Defined MxB mutants influencing GTP binding and hydrolysis revealed that the effector mechanism against herpesviruses is thoroughly different from that against HIV-1. Overall, our findings demonstrate that MxB serves as a broadly acting intracellular restriction factor that controls the establishment of not only retrovirus but also herpesvirus infection of all three subfamilies.

    IMPORTANCE Human herpesviruses pose a constant threat to human health. Reactivation of persisting herpesvirus infections, particularly in immunocompromised individuals and the elderly, can cause severe diseases, such as zoster, pneumonia, encephalitis, or cancer. The interferon system is relevant for the control of herpesvirus replication as exemplified by fatal disease outcomes in patients with primary immunodeficiencies. Here, we describe the interferon-induced, human MX2 gene product MxB as an efficient restriction factor of alpha-, beta-, and gammaherpesviruses. MxB has previously been described as an inhibitor of HIV-1. Importantly, our mutational analyses of MxB reveal an antiviral mechanism of herpesvirus restriction distinct from that against HIV-1. Thus, the dynamin-like MxB GTPase serves as a broadly acting intracellular restriction factor that controls retrovirus as well as herpesvirus infections.

  • Articles of Significant Interest in This Issue [Spotlight]

  • CD8+ T-Cell Response-Associated Evolution of Hepatitis B Virus Core Protein and Disease Progress [Pathogenesis and Immunity]

  • Under the immune pressure of cytotoxic T cells (CTLs), hepatitis B virus (HBV) evolves to accumulate mutations more likely within epitopes to evade immune detection. However, little is known about the specific patterns of the immune pressure-associated HBV mutation of T-cell epitopes and their link to disease progression. Here, we observed a correlation of the accumulated variants on HBV core protein (HBc) with the disease severity of HBV infection. Further analysis indicated that these substitutions were mostly located within CD8+ T-cell epitopes of HBc protein, which were systematically screened and identified in an unbiased manner in our study. From individual peptide level to the human leukocyte antigen I (HLA-I)-restricted population level, we elucidated that the mutations in these well-defined HLA-I-restricted T-cell epitopes significantly decreased antiviral activity-specific CTLs and were positively associated with clinical parameters and disease progression in HBV-infected patients. The molecular pattern for viral epitope variations based on the sequencing of 105 HBV virus genomes indicated that the C-terminal portion (Pc), especially the Pc-1 and Pc-2 positions, have the highest mutation rates. Further structural analysis of HLA-A*02 complexed to diverse CD8+ T-cell epitopes revealed that the highly variable C-terminal bulged peak of M-shaped HBc-derived epitopes are solvent exposed, and most of the CDR3bbeta;s of the T-cell receptor hover over them. These data shed light on the molecular and immunological mechanisms of T-cell immunity-associated viral evolution in hepatitis B progression, which is beneficial for designing immunotherapies and vaccines.

    IMPORTANCE The specific patterns of sequence polymorphisms of T-cell epitopes and the immune mechanisms of the HBV epitope mutation-linked disease progression are largely unclear. In this study, we systematically evaluated the contribution of CD8+ T cells to the disease progress-associated evolution of HBV. By evaluation of patient T-cell responses based on the peptide repertoire, we comprehensively characterized the association of clinical parameters in chronic hepatitis B with the antiviral T-cell response-associated mutations of the viruses from the single-epitope level to the overall HLA-I-restricted peptide levels. Furthermore, we investigated the molecular basis of the HLA-A2-restricted peptide immune escape and found that the solvent-exposed C-terminal portion of the epitopes is highly variable under CDR3bbeta; recognition. Our work may provide a comprehensive evaluation of viral mutations impacted by the host CTL response in HBV disease progression in the context of the full repertoire of HBc-derived epitopes.

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    JVI Accepts: Articles Published Ahead of Print

  • The human cytomegalovirus trimer and pentamer promote sequential steps in entry into epithelial and endothelial cells at cell surfaces and endosomes. [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRaalpha;, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes.

    IMPORTANCE HCMV infects nearly 80 percent of the world's population and causes significant morbidity and mortality. The current anti-viral agents used to treat HCMV infections are prone to resistance and can be toxic to patients and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

  • Transcriptome analysis of infected and bystander type 2 alveolar epithelial cells during influenza A virus infection reveals in vivo Wnt pathway downregulation [PublishAheadOfPrint]

  • Influenza virus outbreaks remain a serious threat to public health. Greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes when compared to AEC from uninfected animals, directly infected cells compared to bystander uninfected AEC produce more interferon and express lower Wnt signaling associated transcripts, while concurrently expressing more transcripts associated with cell death pathways. The Wnt signaling pathway was downregulated in both in vivo infected AEC and in vitro infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.

    IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein expressing influenza virus we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection as compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.

  • In vivo persistence of chimeric virus after substitution of the KSHV LANA DNA binding domain with that of MuHV-4 [PublishAheadOfPrint]

  • The latency associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murine herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice albeit at a lower magnitude than the WT virus. Here, we asked if kLANA can accommodate the mLANA DBD, and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo. Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4.

    IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immune competent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full length kLANA.

  • Infection dynamics of hepatitis E virus in wild-type and immunoglobulin heavy chain knockout JH (-/-) gnotobiotic piglets [PublishAheadOfPrint]

  • Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH (-/-) knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B-lymphocytes in Ig heavy chain JH (-/-) knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH (-/-) knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera when compared to the JH (-/-) knockout pigs, suggesting that the Ig heavy chain JH (-/-) knockout in pigs actually decreased level of HEV replication. Both HEV-infected wild-type and JH (-/-) knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH (-/-) knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight when compared with PBS-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.

    IMPORTANCE According to World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and ggt; 44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH (-/-) knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH (-/-) knockout and wild-type gnotobiotic pig model for HEV, and systemically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH (-/-) knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH (-/-) knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

  • The endonucleolytic RNA cleavage function of nsp1 of Middle East respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type (wt) MERS-CoV and two mutant viruses having specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wt virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wt virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-bbeta; and IFN- mRNAs, however, both mutants were unable to generate intracellular virus particles as efficiently as wt virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.

    IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

  • Sequence variation of Epstein-Barr virus: viral types, geography, codon usage and diseases [PublishAheadOfPrint]

  • 138 new Epstein-Barr virus (EBV) genome sequences have been determined. 125 of these and 116 from previous reports were combined to produce a multiple sequence alignment of 241 EBV genomes, which we have used to analyze variation within the viral genome. The type 1/type2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 SNPs at the EBNA3 locus extend into the adjacent gp350 and gp42 genes, whose products mediate infection of B cells by EBV. A small insertion within the BART miRNA region of the genome was present in 21 EBV strains. EBV from saliva of USA patients with chronic active EBV infection aligned with the wild type EBV genome, with no evidence of WZhet rearrangements. The V3 polymorphism in the Zp promoter for BZLF1 was found to be frequent in nasopharyngeal carcinoma cases both from Hong Kong and Indonesia. Codon usage was found to differ between latent and lytic cycle EBV genes and the main forms of variation of the EBNA1 protein have been identified.

    IMPORTANCE Epstein-Barr virus causes most cases of infectious mononucleosis and post-transplant lymphoproliferative disease. It contributes to several types of cancer including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, nasopharyngeal carcinoma and gastric carcinoma. EBV genome variation is important because some of the diseases associated with EBV have very different incidences in different populations and geographic regions nndash; differences in the EBV genome might contribute to these diseases. Some specific EBV genome alterations that appear to be significant in EBV associated cancers are already known and current efforts to make an EBV vaccine and antiviral drugs should also take account of sequence differences in the proteins used as targets.

  • NEURL3 is an inducible antiviral effector to inhibit HCV assembly by targeting viral E1 glycoprotein [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of Neuralized E3 Ubiquitin Protein Ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection, while had little effect on other RNA viruses including zika virus, dengue virus and vesicular stomatitis virus. Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.

    IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern-recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its NHR domain but not RING domain. Furthermore, we found that NEURL3 has a pan-genotypic anti-HCV activity and interacts with E1 of genotype 2a, 1b, 3a and 6a, but does not inhibit other closely related RNA viruses such as ZIKV, DENV and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.

  • Exploring Crimean-Congo hemorrhagic fever virus-induced hepatic injury using antibody-mediated type I interferon blockade in mice [PublishAheadOfPrint]

  • Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage are poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon signaling (IFN-I) either due to STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h post-exposure to CCHFV, mice developed severe disease with greater than 95% mortality by six days post-exposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days post-exposure and was characterized by hepatocyte necrosis and loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID- (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of TNF superfamily members occurred by day four post-exposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.

    IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small animal systems. The only small animal model in which CCHFV consistently produces severe liver damage are mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a near complete loss of Kupffer cells. Our model system provides insight into both the molecular and cellular features of CCHFV hepatic injury.

  • Using a virion assembly-defective dengue virus as a vaccine approach [PublishAheadOfPrint]

  • Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here we report a virion assembly-defective DENV as a novel vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication, but is completely defective in virion assembly. When trans complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIVNS2A) that can initiate single-round infection. The trans-complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high yield of PIVNS2A production with titers of ggt;107 infectious forming units (IFU)/ml. Mice immunized with a single dose of PIVNS2A elicited strong T cell immune response and neutralization antibodies, and were protected from wild-type virus challenge. Collectively, the results have proved the concept of using assembly-defective virus as a new vaccine approach. The study has also solved the technical bottleneck to produce high yield of PIVNS2A vaccine. The technology could be applicable to vaccine development for other viral pathogens.

    IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we report a novel vaccine platform by using a virion assembly-defective virus. We show that such assembly-defective virus could be rescued to higher titers, and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type virus infection. This vaccine approach could be applicable to other viral pathogens.

  • Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis via the AKT/GSK-3{beta}/{beta}-Catenin Signaling Pathway [PublishAheadOfPrint]

  • Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in hepatitis B virus (HBV) WT-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient mutant-transfected cells and 1.3mer HBV WT-transfected plus reverse transcriptase inhibitor (entecavir or lamivudine) treated cells but all HBV proteins are expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase (GSK-3bbeta;) and increased bbeta;-catenin levels; however, downregulation of Sirt2 in HBV non-replicating cells impaired AKT/GSK-3bbeta;/bbeta;-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activates AKT and consequently increases bbeta;-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by shRNAs or inhibition by AGK2 or dominant-negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased bbeta;-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates bbeta;-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx-independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.

    IMPORTANCE OF THIS STUDY

    Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs via AKT/GSK-3bbeta;/bbeta;-catenin signaling, the relationships between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis are unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication and increases AKT activation, downregulates GSK-3bbeta;, and increases bbeta;-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases bbeta;-catenin. Although HBx activates AKT to upregulate bbeta;-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent development of hepatic fibrosis and HCC.

  • Acute-phase CD4+ T cell responses targeting invariant viral regions are associated with control of live-attenuated simian immunodeficiency virus [PublishAheadOfPrint]

  • We manipulated SIVmac239nef, a model of MHC-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) who express the M3 MHC haplotype that has been associated with poor control of pathogenic SIV. We created SIVnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8+ T cell responses targeting variable epitopes, and that control is achievable in individuals lacking known llsquo;protective' MHC alleles. Full proteome IFN ELISPOT assays identified six newly targeted immunogenic regions following SIVnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVnef-8x targeted at least one of the four invariant regions and had a lower set point viral load compared to two animals that did not target any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8+ T cell responses that target variable regions during SIVmac239nef infection, individuals without llsquo;protective' MHC alleles developed predominantly CD4+ T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4+ T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection.

    IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8+ T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare llsquo;elite controllers', may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is paramount to determine whether T cell responses can be redirected towards invariant viral regions in individuals without llsquo;protective' MHC alleles and if these responses improve control of virus replication.

  • Semen exosomes promote transcriptional silencing of HIV-1 by disrupting NF-kB/Sp1/Tat circuitry [PublishAheadOfPrint]

  • Exosomes play various roles in host responses to cancer and infective agents, and semen exosomes (SE) inhibit HIV-1 infection and transmission, although the mechanism(s) by which this occurs is unclear. Here, we show that SE block HIV-1 proviral transcription at multiple transcriptional check points including transcription factor recruitment to the LTR, and transcription initiation and elongation. Biochemical and functional studies show that SE inhibit HIV-1 long terminal repeat (HIV-LTR)-driven viral gene expression and virus replication. Through partitioning of the HIV-1 RNA, we found that SE reduced the optimal expression of various viral RNA species. CHIP-RT-qPCR and EMSA analysis of infected cells identified human transcription factors NF-kB and Sp1, as well as RNA Pol II and the viral protein Tat as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen PMA or TNFaalpha;. SE inhibited the DNA binding activities of NF-kB and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV LTR promoter. Importantly, SE directly blocked NF-kB, Sp1, and Pol II binding to the LTR, and inhibited the interactions of Tat/NF-kB and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complexmmdash;NF-kB-Sp1-Pol II-Tat, may be a novel mechanism of proviral transcription repression. These data provide a novel molecular basis for SE-mediated inhibition of HIV-1 and identify Tat as a potential target of SE.

    IMPORTANCE HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is need for identifying effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro. In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-kB and Sp1, as well as RNA Pol II to the promoter region in the 5' long terminal repeat (LTR) of HIV-1. The HIV-1 early protein, transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.

  • 5'UTR of human bocavirus capsid transcripts regulates its mRNA biogenesis and alternative translation [PublishAheadOfPrint]

  • The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2 and VP3 in vitro and in vivo. 5' untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, not only affected the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation site (pA)p, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5'UTR of HBoV1 played a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation and progeny virus production.

    IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly and antigenicity. The 5' untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing and they contain different exons. Our study showed that the 5'UTR not only modulated mRNA abundance but also regulated capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5'UTR were found to affect viral capsid mRNA polyadenylation, alternative translation and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.

  • Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase I aalpha; (CKIaalpha;). In line with this, alanine mutation at S225, S229, or S232 reduced whereas aspartate mutation at the same sites rescued NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIaalpha; inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIaalpha;-mediated intramolecular sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.

    IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence is missing. We offered the first direct evidence of CKIaalpha;-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.

  • Substrate binding by the 2nd sialic acid-binding site of influenza A virus N1 neuraminidase contributes to enzymatic activity [PublishAheadOfPrint]

  • The influenza A virus (IAV) neuraminidase (NA) protein plays an essential role in the release of virus particles from cells and decoy receptors. The NA enzymatic activity presumably needs to match the activity of the IAV hemagglutinin (HA) attachment protein and the host sialic acid (SIA) receptor repertoire. We analyzed the enzymatic activities of N1 NA proteins derived from avian (H5N1) and human (H1N1) IAVs and analyzed the role of the 2nd SIA-binding site, located adjacent to the conserved catalytic site, therein. SIA-contact residues in the 2nd SIA-binding site of NA are highly conserved in avian, but not in human IAVs. All N1 proteins preferred cleaving aalpha;2,3- over aalpha;2,6-linked SIAs, even when their corresponding HA protein displayed a strict preference for aalpha;2,6-linked SIAs, indicating that the specificity of the NA protein does not need to fully match that of the corresponding HA protein. NA activity was affected by substitutions in the 2nd SIA-binding site that are observed in avian and human IAVs, at least when multivalent rather than monovalent substrates were used. These mutations included both SIA-contact residues and residues that do not directly interact with SIA in all three loops of the 2nd SIA-binding site. Substrate binding via the 2nd SIA-binding site enhanced the catalytic activity of N1. Mutation of the 2nd SIA-binding site was also shown to affect virus replication in vitro. Our results indicate an important role for the N1 2nd SIA-binding site in binding to and cleavage of multivalent substrates.

    Significance

    Avian and human influenza A viruses (IAVs) preferentially bind aalpha;2,3- or aalpha;2,6-linked sialic acids (SIAs), respectively. A functional balance between the hemagglutinin (HA) attachment and neuraminidase (NA) proteins is thought to be important for host tropism. What this balance entails at the molecular level is, however, not well understood. We now show that N1 proteins of both avian and human viruses prefer cleaving avian- over human-type receptors, although human viruses were relatively better in cleavage of the human-type receptors. In addition, we show that substitutions at different positions in the 2nd SIA-binding site found in NA proteins of human IAVs have a profound effect on binding and cleavage of multivalent, but not monovalent, receptors and affect virus replication. Our results indicate that the HA-NA balance can be tuned via modification of substrate binding via this site and suggest an important role of the 2nd SIA-binding site in host tropism.

  • Temperature-dependent nanomechanics and topography of bacteriophage T7 [PublishAheadOfPrint]

  • Viruses are nanoscale infectious agents, which may be inactivated by heat treatment. The global molecular mechanisms of virus inactivation and the thermally-induced structural changes in viruses are not fully understood. Here we measured the heat-induced changes in the properties of T7 bacteriophage particles exposed to two-stage (65 ddeg;C and 80 ddeg;C) thermal effect, by using AFM-based nanomechanical and topographical measurements. We found that exposure to 65 ddeg;C led to the release of genomic DNA and to the loss of the capsid tail, hence the T7 particles became destabilized. Further heating to 80 ddeg;C surprisingly led to an increase in mechanical stability, due likely to partial denaturation of the capsomeric proteins kept within the global capsid arrangement.

    IMPORTANCE Even though the loss of DNA, caused by heat treatment, destabilizes the T7 phage, its capsid is remarkably able to withstand high temperatures with a more-or-less intact global topographical structure. Thus, partial denaturation within the global structural constraints of the viral capsid may have a stabilizing effect. Understanding the structural design of viruses may help in constructing artificial nanocapsules for the packaging and delivery of materials under harsh environmental conditions.

  • Association between microRNA-373 and long non-coding RNA NORAD in hepatitis C virus infected hepatocytes impairs Wee1 expression for growth promotion [PublishAheadOfPrint]

  • Chronic hepatitis C virus (HCV) infection may lead to end stage liver disease, including hepatocellular carcinoma (HCC). We have previously shown that microRNA-373 (miR-373) is upregulated in HCV infected human liver biopsy specimens. To gain insights the role of miR-373 in HCV mediated pathogenesis, we investigated its interacting partner for hepatocyte growth regulation. RNA-seq data revealed that Wee1 is associated with miR-373, and is a direct target. Interestingly, higher expression of Wee1 was noted in HCV infected hepatocytes, suggesting other factors may block miR-373 mediated Wee1 inhibition. We subsequently found an association between long noncoding RNA NORAD (LINC000657) and miR-373, and demonstrated that NORAD binds to miR-373 and Wee1 independently. However, higher Wee1 expression in HCV infected hepatocytes suggested that miR-373 form complex with NORAD. Depletion of miR-373 or Wee1 inhibitor reduces the HCV genome harbouring Huh7.5 cell growth and Wee1 expression. Together, our study demonstrates a novel mechanism of hepatocyte growth promotion during HCV infection involving miR-373-NORAD-Wee1 axis, and may be a target for future therapy against HCV associated HCC.

    IMPORTANCE Mechanism of HCV mediated liver pathogenesis is poorly understood. In this study, we observed that HCV infection upregulates miR-373 and Wee1, a pivotal player in G2 checkpoint in cell cycle, although Wee1 is direct target for miR-373. Our subsequent study demonstrated that miR-373 form complex with long noncoding RNA NORAD, resulting in release of their common target Wee1 in HCV infected cells, which in turn favours uncontrolled cell growth. Our study suggested a previously unknown mechanism for hepatocyte growth promotion following HCV infection, and this pathway can be targeted for future therapy against HCV mediated liver pathogenesis.

  • Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-zoster Virus [PublishAheadOfPrint]

  • Both type I and type II interferons (IFNs) have been implicated in the host defense against varicella zoster virus (VZV), a common human herpesvirus that causes varicella and zoster. The purpose of this study was to compare their contributions to the control of VZV replication, to identify the signaling pathways that are critical for mediating their antiviral activity, and to define mechanisms by which the virus counteracts their effects. IFN was much more potent than IFNaalpha; in blocking VZV infection, which was associated with a differential induction of the interferon regulatory factor (IRF) proteins, IRF1 and IRF9, respectively. These observations account for the clinical experience that while the formation of VZV skin lesions is controlled initially by local immunity, adaptive virus-specific T cell responses are required to prevent life-threatening VZV infections.

    Importance

    While both Type I and Type II IFNs are involved in the control of herpesvirus infections in the human host, to our knowledge, their relative contributions to the restriction of viral replication and spread have not been assessed. We report that IFN has more potent activity than IFNaalpha; against VZV. Findings from this comparative analysis show that the IFNaalpha;-IRF9 axis functions as a first line of defense to delay the onset of viral replication and spread whereas the IFN-IRF1 axis has the capacity to block the infectious process. Our findings underscore the importance of IRFs in IFN regulation of herpesvirus infection and account for the clinical experience of initial control of VZV skin infection, attributable to IFNaalpha; production, together the requirement for induction of adaptive IFN-producing VZV specific T cells to resolve the infection.

  • Structural and Functional Characterization of Membrane Fusion Inhibitors with Extremely Potent Activity against HIV-1, HIV-2, and Simian Immunodeficiency Virus [PublishAheadOfPrint]

  • T-20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it has low anti-HIV activity and genetic barrier for drug resistance. We recently reported that T-20 sequence-based lipopeptides possess extremely potent in vitro and in vivo efficacies (Ding et al. J Virol, 2017; Chong et al. J Virol, 2018). Herein, we focused on characterizing the structure-activity relationships of the T-20 derivatives. First, a novel lipopeptide termed LP-52 was generated with improved target-binding stability and anti-HIV activity. Second, a large panel of truncated lipopeptides were characterized, revealing a 21-amino acid sequence core structure. Third, it was surprisingly found that addition of the gp41 pocket-binding residues in the N-terminus of new inhibitors resulted in increased binding but decreased antiviral activities. Fourth, while LP-52 showed the most potent activity in inhibiting divergent HIV-1 subtypes, its truncated versions such as LP-55 (25-mer) and LP-65 (24-mer) still maintained their potencies at very low picomolar concentrations (pM); however, both the N- and C-terminal motifs of LP-52 played crucial roles on the inhibition of T-20-resistant HIV-1 mutants, HIV-2, and SIV isolates. Fifth, we verified that LP-52 can bind to target cell membranes and human serum albumin, and has low cytotoxicity and a high genetic barrier to inducing drug resistance.

    IMPORTANCE Development of novel membrane fusion inhibitors against HIV and other enveloped viruses is highly important in terms of the peptide drug T-20 remains the only one for clinical use even if it is limited by large dosages and resistance. Here, we report a novel T-20 sequence-based lipopeptide showing extremely potent and broad activities against HIV-1, HIV-2, SIV, and T-20-resistant mutants as well as extremely high therapeutic selectivity index and genetic resistance barrier. The structure-activity relationship (SAR) of the T-20 derivatives has been comprehensively characterized, revealing a critical sequence core structure and the target sites of viral vulnerability that not include the gp41 pocket. The results also suggest that membrane-anchored inhibitors possess unique modes of action relative to unconjugated peptides. Combined, our series studies have not only provided drug candidates for clinical development but also offered important tools to elucidate the mechanisms of viral fusion and inhibition.

  • Assembly properties of Hepatitis B Virus core protein mutants correlate with their resistance to assembly-directed antivirals [PublishAheadOfPrint]

  • Hepatitis B Virus (HBV) capsid or core protein (Cp) can self-assemble to form an icosahedral capsid. It is now pursued as a target for small molecule antivirals that enhance the rate and extent of its assembly to yield empty and/or aberrant capsids. These small molecules are thus called Core protein Allosteric Modulators (CpAMs). We sought to understand the physical basis of CpAM-resistant mutants and how CpAMs might overcome them. We examined the effects of two closely related CpAMs, HAP12 and HAP13, which differ by a single atom but have drastically different antiviral activity, on the assembly of wildtype Cp and three T109 mutants (T109M, I, S) that display a range of resistance. The T109 side chain forms part of the mouth of the CpAM-binding pocket. A T109 mutant that has substantial resistance even to a highly active CpAM strongly promotes normal assembly. Conversely, a mutant that weakens assembly is more susceptible to CpAMs. In crystal and cryo-electron microscopy (cryo-EM) structures of T=4 capsids with bound CpAMs, the CpAMs preferentially fit into two of four quasi-equivalent sites. In these static representations of capsid structures, T109 does not interact with the neighboring subunit. However, all-atom molecular dynamics simulations of an intact capsid show that T109 of one of the four classes of CpAM site has a hydrophobic contact with the neighboring subunit at least 40% of the time, providing a physical explanation for the mutation's ability to affect capsid stability, assembly, and sensitivity to CpAMs.

    Importance

    The HBV core protein and its assembly into capsids have become important targets for development of core protein allosteric modulators (CpAMs) as antivirals. Naturally occurring T109 mutants have been shown to be resistant to some of these CpAMs. We found that mutation of T109 led to changes in capsid stability and recapitulated resistance to a weak CpAM but much less so to a strong CpAM. Examination of HBV capsid structures, determined by cryo-EM and crystallography, could not explain how T109 mutations change capsid stability and resistance. However, by mining data from a long-duration all-atom molecular dynamics simulation we found that the capsid was extraordinarily flexible and that T109 can impede entry to the CpAM binding site. In short, HBV capsids are incredibly dynamic and that molecular mobility must be considered when discussing antiviral mechanism.

  • Adjuvant-dependent enhancement of HIV Env-specific antibody responses in infant rhesus macaques [PublishAheadOfPrint]

  • Towards the goal of developing an effective HIV vaccine that can be administered in infancy to protect against postnatal and life-long sexual HIV transmission risks, the current pilot study was designed to compare the effect of novel adjuvants on the induction of HIV Env-specific antibody responses in infant macaques. Aligning our studies with the adjuvanted proteins evaluated in a prime-boost schedule with ALVAC in the ongoing HVTN 702 efficacy trial, we selected the bivalent clade C Env immunogens gp120 C.1086 and gp120 TV1 in combination with the MF59 adjuvant. However, we hypothesized that the Adjuvant System AS01, that is included in the pediatric RTS,S malaria vaccine, would promote superior Env-specific antibody responses compared to the oil-in-water MF59 emulsion adjuvant. In a second study arm, we compared two emulsions, GLA-SE and 3M-052-SE, containing a TLR4 or TLR7/8 ligand, respectively. The latter adjuvant had been previously demonstrated to be especially effective in activating neonatal antigen-presenting cells.

    Our results demonstrate that different adjuvants drive a quantitatively or qualitatively distinct response to the bivalent Env vaccine. AS01 induced higher Env-specific plasma IgG antibody levels and promoted improved antibody function compared to infants that received the antigen in MF59, and 3M-052-SE outperformed GLA-SE by inducing the highest breadth and functionality of antibody responses.

    Thus, distinct adjuvants are likely to be required for maximizing vaccine-elicited immune responses in infants, particularly when immunization in infancy aims to elicit both perinatal and life-long immunity against challenging pathogens such as HIV.

    IMPORTANCE

    Alum remains the adjuvant of choice for pediatric vaccines. Yet, the distinct nature of the developing immune system in infants likely requires novel adjuvants targeted specifically at the pediatric population to reach maximal vaccine efficacy with an acceptable safety profile.

    The current study supports the idea that additional adjuvants for pediatric vaccines should and need to be tested in infants for their potential to enhance immune responses. Using an infant macaque model, our results suggest that both AS01 and 3M-052-SE can significantly improve and better sustain HIV Env-specific antibody responses compared to alum. Despite the limited number of animals, the results revealed interesting differences that warrant further testing of promising novel adjuvants candidates in larger preclinical and clinical studies to define the mechanisms leading to adjuvant-improved antibody responses and to identify targets for adjuvant and vaccine optimization.

  • Mapping an adeno-associated virus 9-specific neutralizing epitope to develop next-generation gene delivery vectors [PublishAheadOfPrint]

  • Recent clinical trials have demonstrated the potential of adeno-associated virus (AAV)-based vectors for treating rare diseases. However, significant barriers remain for the translation of these vectors into widely available therapies. In particular, exposure to the AAV capsid can generate an immune response of neutralizing antibodies. One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization. To accomplish this, we isolated a monoclonal antibody against AAV9 following immunization of Balb/c mice and hybridoma screening. This antibody, PAV9.1, is specific for intact AAV9 capsids and has a high neutralizing titer of ggt;1:160,000. We used cryo-electron microscopy to reconstruct PAV9.1 in complex with AAV9. We then mapped its epitope to the threefold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588-QAQAQT-592. Capsid mutagenesis demonstrated that even a single amino acid substitution within this epitope markedly reduced binding and neutralization by PAV9.1. Additionally, in vivo studies showed that mutations in the PAV9.1 epitope conferred a "liver-detargeting" phenotype to the mutant vectors, unlike AAV9, indicating that the residues involved in PAV9.1 interactions are also responsible for AAV9 tropism. However, we observed minimal changes in binding and neutralizing titer when we tested these mutant vectors for evasion of polyclonal sera from mice, macaques, or humans previously exposed to AAV. Taken together, these studies demonstrate the complexity of incorporating mapped neutralizing epitopes and previously identified functional motifs into the design of novel capsids able to evade immune response.

    IMPORTANCE Gene therapy utilizing viral vectors has experienced recent success, culminating in Food and Drug Administration approval of the first adeno-associated virus vector gene therapy product in the United States: Luxturna for inherited retinal dystrophy. However, application of this approach to other tissues faces significant barriers. One challenge is the immune response to viral infection or vector administration, precluding patients from receiving an initial or readministered dose of vector, respectively. Here, we mapped the epitope of a novel neutralizing antibody generated in response to this viral vector to design a next-generation capsid to evade immune responses. Epitope-based mutations in the capsid interfered with the binding and neutralizing ability of the antibody but not when tested against polyclonal samples from various sources. Our results suggest that targeted mutation of a greater breadth of neutralizing epitopes will be required to evade the repertoire of neutralizing antibodies responsible for blocking viral vector transduction.

  • Intrahepatic Cross-Presentation and Hepatocellular Antigen Presentation Play Distinct Roles in the Induction of HBV-Specific CD8+ T Cell Responses [PublishAheadOfPrint]

  • CD8+ T cells are the key cellular effectors mediating the clearance of hepatitis B virus (HBV) infections. However, early immunological events surrounding the priming of HBV-specific CD8+ T cell responses remain poorly understood. This study examined the importance of priming location and the relative contribution of endogenous antigen presentation by hepatocytes versus cross-presentation by bone-marrow derived cells to the induction of functional HBV-specific CD8+ T cell responses using the animal models of acute and chronic HBV infection. Functional HBV-specific CD8+ T cell responses could be induced to intrahepatically expressed HBV even when T cell homing to the lymphoid tissues was severely suppressed, suggesting that functional priming could occur in the liver. The expansion of HBV-specific CD8+ T cells was significantly reduced in the mice whose MHC class I expression was mostly restricted to non-hematopoietic cells, suggesting the importance of cross-presentation by hematopoietic cells in the induction of HBV-specific CD8+ T cells. Strikingly, the expansion and cytolytic differentiation of HBV-specific CD8+ T cells were reduced even more severely in the mice whose MHC class I expression was restricted to hematopoietic cells. Collectively, these results indicate that cross-presentation is required, but relatively inefficient in terms of inducing the cytolytic differentiation of HBV-specific CD8+ T cells by itself. Instead, the expansion and functional differentiation of HBV-specific CD8+ T cells are primarily dependent on hepatocellular antigen presentation.

    IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic hepatitis. Approximately 260 million people are chronically infected with HBV and under an increased risk of developing cirrhosis and hepatocellular carcinoma. Host immune responses, particularly HBV-specific CD8+ T cell responses, largely determine the outcome of HBV infection. It is widely accepted that antigen inexperienced CD8+ T cells should be initially activated by professional antigen presenting cells (pAPCs) in lymphoid tissues to differentiate into effector CD8+ T cells. However, this notion has not been tested for HBV-specific CD8+ T cells. In this study, we show that HBV-specific CD8+ T cell responses can be induced in the liver. Surprisingly, antigen presentation by hepatocytes is more important than cross-presentation by hematopoietic cells for the induction of HBV- specific CD8+ T cell responses. These results revealed a previously unappreciated role of antigen presentation by hepatocytes in the induction of HBV-specific CD8+ T cell responses.

  • Human Cytomegalovirus UL135 Interacts with Host Adaptor Proteins to Regulate Epidermal Growth Factor Receptor and Reactivation from Latency [PublishAheadOfPrint]

  • Human cytomegalovirus, HCMV, is a beta-herpesvirus that establishes a life-long latent infection in its host that is marked by recurrent episodes of reactivation. The molecular mechanisms by which the virus and host regulate entry into and exit from latency remain poorly understood. We have previously reported that UL135 is critical for reactivation, functioning in part by overcoming suppressive effects of the latency determinant UL138. We have demonstrated a role for UL135 in diminishing cell surface levels and inhibiting the return of epidermal growth factor receptor (EGFR) to the cell surface. The attenuation of EGFR signaling promotes HCMV reactivation in combination with cellular differentiation. In this study, we sought to define the mechanisms by which UL135 functions in regulating EGFR turnover and viral latency. Screens to identify proteins interacting with pUL135 identified two host adaptor proteins, CIN85 and Abi-1, with overlapping activities in regulating EGFR levels in the cell. We mapped the amino acids in pUL135 necessary for interaction with Abi-1 and CIN85, and generated recombinant viruses expressing variants of pUL135 that do not interact with CIN85 or Abi-1. These recombinant viruses replicate in fibroblasts but are defective for reactivation in an experimental model for latency using primary CD34+ hematopoietic progenitor cells (HPCs). These UL135 variants have altered trafficking of EGFR and are defective in targeting EGFR for turnover. These studies demonstrate a requirement for pUL135 interactions with Abi-1 and CIN85 for regulation of EGFR and mechanistically link the regulation of EGFR to reactivation.

    Importance

    Human cytomegalovirus (HCMV) establishes a life-long latent infection in the human host. While the infection is typically asymptomatic in healthy individuals, HCMV infection poses life-threatening disease risk in immunocompromised individuals and is the leading cause of birth defects. Understanding how HCMV controls the life-long latent infection and reactivation of replication from latency is critical to developing strategies to control HCMV disease. Here, we identify the host factors targeted by a viral protein that is required for reactivation. We define the importance of this virus-host interaction in reactivation from latency, providing new insights into the molecular underpinnings of HCMV latency and reactivation.

  • Selective 4-thiouracil labeling of RNA transcripts within latently infected cells after infection with human cytomegalovirus expressing functional uracil phosphoribosyltransferase [PublishAheadOfPrint]

  • Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent lifecycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. As only a small fraction of these cell types harbor viral genomes during natural latency, the identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Herein, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.

    IMPORTANCE

    HCMV is a significant pathogen that accounts for a substantial amount of complications within the immuno-suppressed and immuno-compromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation post-transplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.

  • PF74 reinforces the HIV-1 capsid to impair reverse transcription-induced uncoating [PublishAheadOfPrint]

  • The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here we show, using atomic force microscopy, that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilzes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 mmu;M, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure observed to form during reverse transcription in vitro persisted throughout the entire duration of the measurement (~24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription yet interfering with a late step in uncoating.

    IMPORTANCE The capsid binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physica properties of HIV-1 cores during the course of reverse transcription. Here we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociates from the narrow end of the core HIV-1. Our result provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.

  • Epstein-Barr virus type-2 infects T-cells and induces B-cell lymphomagenesis in humanized mice [PublishAheadOfPrint]

  • Epstein Barr virus (EBV) has been classified into two strains, EBV Type-1 (EBV-1) and EBV Type-2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B-cells in culture while EBV-2 is poorly transforming. The differing ability to immortalize B-cells in vitro suggests that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T-cells, infecting T-cells in culture and in healthy Kenyan infants, strongly suggesting EBV-2 infection of T-cells is a natural part of the EBV-2 life-cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T-cells. Therefore, BALB/c-Rag2null IL2rnull SIRPaalpha; humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T- and B-cells, unlike infection with EBV-1, in which only B-cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B- and T-cells. Importantly, EBV-2 infected mice developed tumors resembling diffuse large B-cell lymphoma (DLBCL). These lymphomas had morphological features comparable to EBV-1 induced DLBCL, developed at similar rates with equivalent frequency, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B-cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T-cells, and the capacity of EBV-2 to drive lymphomagenesis.

    IMPORTANCE EBV is a well-established B-cell tropic virus. However, we have recently shown that the EBV Type 2 (EBV-2) strain also infects primary T-cells in culture and in healthy Kenyan children. This finding suggests that EBV-2- unlike the well-studied EBV-1 strain, utilizes the T-cell compartment to persist. As EBV is human-specific, studies to understand the role of T-cells in EBV-2 persistence requires an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34+ stem cells. Characterization of the EBV-2 infected humanized mice established that both T-cells and B-cells are infected by EBV-2 and the majority of infected mice develop a B-cell lymphoma resembling diffuse large B-cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T-cells contributes to persistence and lymphomagenesis.

  • Discovering Invisible Truths [PublishAheadOfPrint]

  • The rise of populist movements worldwide is challenging science and motivating scientists to join the debate and enter politics. Based on my experience, taking a public stand will not come without slanderous personal and institutional attacks as an attempt to shake scientific credibility. The virology community is at risk of similar misrepresentation; reflection on this topic, and particularly on how to address such challenges, should be a priority, given we are in the "post-truth" era.

  • Kinesin-1 proteins KIF5A, 5B and 5C promote anterograde transport of herpes simplex virus enveloped virions in axons. [PublishAheadOfPrint]

  • Herpes simplex virus and other aalpha;-herpesviruses must spread from sites of viral latency in sensory ganglia to peripheral tissues where the viruses can replicate to higher titers then spread to other hosts. These viruses move in neuronal axons from ganglia to the periphery propelled by kinesin motors moving along microtubules. Two forms of HSV particles undergo this anterograde transport in axons: i) unenveloped capsids that become enveloped after reaching axon tips and ii) enveloped virions that are transported within membrane vesicles in axons. Fundamental toward understanding this axonal transport is the question of which of many different axonal kinesins convey HSV particles. Knowing which kinesins promote axonal transport would provide clues to the identity of HSV proteins that tether onto kinesins. Prominent among axonal kinesins are the kinesin-1 (KIF5A, B, C) and kinesin-3 (e.g. KIF1A, 1B) families. We characterized fluorescent forms of cellular cargo molecules to determine if enveloped HSV particles were present in the vesicles containing these cargos. Kinesin-1 cargo proteins were present in vesicles containing HSV particles, but not kinesin-3 cargos. Fluorescent kinesin-1 protein KIF5C extensively colocalized with HSV particles, while fluorescent kinesin- 1 KIF1A did not. Silencing of kinesin-1 proteins KIF5A, 5B, and 5C or light chains KLC1 and KLC2 inhibited the majority of HSV anterograde transport, while silencing of KIF1A had little effect on HSV transport in axons. We concluded that kinesin-1 proteins are important in the anterograde transport of the majority of HSV enveloped virions in neuronal axons and kinesin-3 proteins are less important.

    Importance

    Herpes simplex virus (HSV) and other aalpha;-herpesviruses such as varicella zoster virus depend upon the capacity to navigate in neuronal axons. To do this virus particles tether onto dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde) or from cell bodies to axon tips (anterograde). Following reactivation from latency, aalpha;-herpesvirus absolutely depend upon anterograde transport of virus particles in axons in order that the viruses can reinfect peripheral tissues and spread to other hosts. Which of the many axonal kinesins transport HSV in axons is not clear. We characterized fluorescent cellular cargo molecules and kinesins to provide evidence that HSV enveloped particles are ferried by kinesin-1 proteins KIF5A, 5B and 5C and their light chains, KLC1 and KLC2 in axons. Moreover, we provided evidence that kinesin-1 proteins are functionally important in anterograde transport of HSV virions by silencing these proteins.

  • Epstein Barr Virus-Induced Nodules on Viral Replication Compartments Contain RNA Processing Proteins and a Viral Long Non-Coding RNA [PublishAheadOfPrint]

  • Profound alterations in host cell nuclear architecture accompany the lytic phase of Epstein-Barr virus (EBV) infection. Viral replication compartments assemble; host chromatin marginalizes to the nuclear periphery; cytoplasmic poly(A)-binding protein translocates to the nucleus; polyadenylated mRNAs are sequestered within the nucleus. Virus-induced changes to nuclear architecture that contribute to viral host shutoff (vhs) must accommodate selective processing and export of viral mRNAs. Here we describe additional previously unrecognized nuclear alterations during EBV lytic infection in which viral and cellular factors that function in pre-mRNA processing and mRNA export are redistributed. Early during lytic infection, before formation of viral replication compartments, two cellular pre-mRNA splicing factors, SC35 and SON, were dispersed from interchromatin granule clusters, and three mRNA export factors, Y14, ALY, and NXF1, were depleted from the nucleus. During late lytic infection, virus-induced nodular structures (VINORCs) formed at the periphery of viral replication compartments. VINORCs were composed of viral (BMLF1 and BGLF5) and cellular (SC35, SON, SRp20, NXF1) proteins that mediate pre-mRNA processing and mRNA export. BHLF1 long non-coding RNA was invariably found in VINORCs. VINORCs did not contain other nodular nuclear cellular proteins (PML, coilin), nor did they contain viral proteins (BRLF1, BMRF1) found exclusively within replication compartments. VINORCs are novel EBV-induced nuclear structures. We propose that EBV-induced dispersal and depletion of pre-mRNA processing and mRNA export factors during early lytic infection contribute to vhs; subsequent relocalization of these pre-mRNA processing and mRNA export proteins to VINORCs and viral replication compartments facilitate selective processing and export of viral mRNAs.

    IMPORTANCE

    In order to make protein, mRNA transcribed from DNA in the nucleus must enter the cytoplasm. Nuclear export of mRNA requires correct processing of mRNAs by enzymes that function in splicing and nuclear export. During the Epstein Barr virus (EBV) lytic cycle, nuclear export of cellular mRNAs is blocked, yet export of viral mRNAs is facilitated. In this study we report the dispersal and dramatic reorganization of cellular (SC35, SON, SRp20, Y14, ALY, NXF1) and viral (BMLF1, BGLF5) proteins that play key roles in pre-mRNA processing and export of mRNA. These virus-induced nuclear changes culminate in formation of VINORCs, novel nodular structures composed of viral and cellular RNA splicing and export factors. VINORCs localize to the periphery of viral replication compartments where viral mRNAs reside. These EBV-induced changes in nuclear organization may contribute to blockade of nuclear export of host mRNA, while enabling selective processing and export of viral mRNA.

  • HIV-1 employs multiple mechanisms to resist Cas9/sgRNA targeting the viral primer binding site [PublishAheadOfPrint]

  • The CRISPR/Cas9 gene editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections including HIV-1. This utility of CRISPR/Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of single guide RNA (sgRNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS) that HIV-1 copies from cellular tRNALys.3 in each round of reverse transcription, and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes indels, and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms informs future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.

    IMPORTANCE

    Results of this study demonstrate that the gene editing complex Cas9/sgRNA can be programmed to target and cleave the HIV-1 PBS DNA, thus inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS which is copied from the cellular tRNALys.3 during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target either the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant, which uses the "CCA" sequence as the PAM (protospacer adjacent motif), should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

  • Contribution of a multifunctional polymerase region of foot-and-mouth disease virus to lethal mutagenesis [PublishAheadOfPrint]

  • Viral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays similar polymerase activity, RNA binding, and copying processivity than the wild type enzyme, but increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop bbeta;9-aalpha;11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed.

    IMPORTANCE The nuclear localization signal (NLS) of the foot-and-mouth disease virus (FMDV) polymerase includes residues that modulate the sensitivity to mutagenic agents. Here we have described a viable NLS mutant with an amino acid replacement that facilitates virus extinction by ribavirin. The corresponding polymerase shows increased incorporation of ribavirin-triphosphate, and local structural modifications that implicate the template entry channel. Specifically, comparison of the structure of ribavirin-sensitive and ribavirin-resistant FMDV polymerases has identified loop bbeta;9-aalpha;11 conformation as determinant of sensitivity to ribavirin mutagenesis.

  • Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL [PublishAheadOfPrint]

  • Zika virus (ZIKV) is genetically and biologically related to other Flaviviridae family members and has disseminated to many countries. It is associated with severe consequences including the abnormal development of the neural system in fetuses and neurological diseases in adults. Therefore, developing anti-ZIKV drugs is of paramount importance. Screening of generic drugs revealed that several non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, acetaminophen, and lornoxicam potently inhibit the entry of Zika-Env/HIV-1 pseudotyped viruses. They also significantly inhibited the replication of wildtype ZIKV, both in cell lines and in primary human fetal endothelial cells. Interestingly, NSAIDs exerted this inhibitory effect by potently reducing the expression of AXL, the entry cofactor of ZIKV. Further studies showed that NSAIDs down-regulated the PGE2/EP/cAMP/PKA signaling pathway, and reduced PKA-dependent CDC37 phosphorylation and the interaction between CDC37 and HSP90, which subsequently facilitated the CHIP/ubiquitination/proteasome-mediated AXL degradation. Taken together, our results highlight a new mechanism of action of antiviral agents, which may assist in designing a convenient strategy for treating ZIKV-infected patients.

    IMPORTANCE Zika virus (ZIKV) infection, which causes congenital malformations, including microcephaly and other neurological disorders, has attracted global attention. We observed that several NSAIDs significantly inhibited ZIKV infection. Based on our observations, we proposed a novel mechanism of action of antiviral compounds, which involves blockade of virus entry via degradation of the entry cofactor. Furthermore, NSAIDs can be practically used for preventing ZIKV infection in pregnant women, as certain NSAIDs, including ibuprofen and acetaminophen, are considered clinically safe.

  • Potato spindle tuber viroid modulates its replication through a direct interaction with a splicing regulator [PublishAheadOfPrint]

  • Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic make-up and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II (Pol II) for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5; RPL5) in vitro and in vivo. PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions.

    Importance

    Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants often resulting in disease. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA-7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control.

  • Complement C3 Plays a Key Role in Inducing Humoral and Cellular Immune Responses to Influenza Virus Strain Specific HA or Cross Protective M2e vaccination. [PublishAheadOfPrint]

  • The complement pathway is involved in eliminating antigen immune complexes. However, the role of the C3 complement system remains largely unknown in influenza virus M2 extracellular (M2e) domain or hemagglutinin (HA) vaccine-mediated protection after vaccination. Using a C3 knockout (C3 KO) mouse model, we found that complement protein C3 was required for effective induction of immune responses to vaccination with M2e-based or HA-based vaccines, which include isotype class-switched antibodies and effector CD4 and CD8 T cell responses. C3 KO mice after active immunization with cross protective non-neutralizing M2e-based vaccine were not protected against influenza virus although low levels of M2e specific antibodies were protective after passive co-administration with virus in wild type mice. In contrast, C3 KO mice that were immunized with strain-specific neutralizing HA-based vaccine were protected against homologous virus challenge despite lower levels of HA antibody responses. C3 KO mice showed impaired maintenance of innate immune cells and a defect in innate immune responses upon exposure to antigens. The findings in this study suggest that C3 is required for effective induction of humoral and cellular adaptive immune responses as well as protective immunity after non-neutralizing influenza M2e vaccination.

    IMPORTANCE

    Complement is the well-known innate immune defense system by involving in the opsonization and lysis of pathogens but less studied in establishing adaptive immunity after vaccination. Influenza virus HA-based vaccination confers protection via strain-specific neutralizing antibodies whereas M2e-based vaccination induces a broad spectrum of protection by immunity against the conserved M2e epitopes. This study revealed the critical roles of C3 complement in inducing humoral and cellular immune responses after immunization with M2e or HA vaccines. C3 was found to be required for protection by M2e-based but not by HA-based active vaccination as well as for maintaining innate antigen presenting cells. Findings in this study have insight into better understanding the roles of C3 complement in inducing effective innate and adaptive immunity as well as in conferring protection by cross protective conserved M2e vaccination.

  • Functional study of the C-terminal part of hepatitis C virus E1 ectodomain [PublishAheadOfPrint]

  • In the HCV envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acids (aa) 270 and 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the aa314-342 region, which has been shown to contain two alpha helices (aalpha;2 and aalpha;3) by NMR studies. Twenty out of the twenty-two generated mutants were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI co-receptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the aalpha;-helix 315-324 residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its co-receptors and in HCV assembly. They also highlight the involvement of aalpha;-helix 315-324 in a late step of HCV entry.

    Importance: HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. Here we characterized the function of the putative fusion peptide and the region containing alpha helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the aalpha;2 helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

  • Inhibition of stress granule formation by Middle East respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication [PublishAheadOfPrint]

  • Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. MERS-CoV 4a accessory protein is known to inhibit SG formation in expressed cells by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated eIF2aalpha; phosphorylation. Replication of MERS-CoV lacking genes 4a and 4b (MERS-CoV-p4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-p4-induced SG formation. MERS-CoV-p4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells and inhibition of SG formation by siRNA-mediated depletion of the SG components promoted MERS-CoV-p4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-p4 replication was neither due to induction of type I and type III interferons nor accumulation of viral mRNAs in the SGs. Rather, it was due to inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2aalpha; phosphorylation and likely by confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.

    IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that MERS-CoV 4a accessory protein, which was reported to block SG formation in expressed cells, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this is the first report showing the biological significance of SG in CoV replication, and provides insight into the interplay between MERS-CoV and antiviral stress responses.

  • Protein mismatches caused by reassortment influence functions of the reovirus capsid [PublishAheadOfPrint]

  • Following attachment to host receptors via 1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing 1 and membrane-targeting peptides from the viral mmu;1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3DF/T3DCS1, a S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3DF and T3DC. T3DF/T3DCS1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded 1 on T3DF/T3DCS1 virions. Additionally, in comparison to T3DF, T3DF/T3DCS1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for 1 in regulating ISVP* formation. The 1 protein is held within turrets formed by the 2 protein. To test if the altered properties of T3DF/T3DCS1 are due to a mismatch between 1 and 2 proteins from T3DF and T3DC, properties of T3DF/T3DCL2 and T3DF/T3DCS1L2, which express a T3DC-derived 2, were compared. The presence of T3DC 2 allowed more efficient 1 incorporation, producing particles that exhibit T3DF-like infectivity. In comparison to T3DF, T3DF/T3DCL2 prematurely converts to ISVP* uncovering a role for 2 in regulating ISVP* formation. Importantly, a virus with matching 1 and 2 displayed a more regulated conversion to ISVP* than either T3DF/T3DCS1 or T3DF/T3DCL2. In addition to identifying new regulators of ISVP* formation, our results highlight that protein mismatches produced by reassortment can alter virus assembly and thereby influence subsequent functions of the virus capsid.

    IMPORTANCE Cells coinfected with viruses that possess a multipartite or segmented genome reassort to produce progeny viruses that contain a combination of gene segments from each parent. Reassortment places new pairs of genes together generating viruses in which mismatched proteins must function together. To test if such forced pairing of proteins that form the virus shell or capsid alters the function of the particle, we investigated properties of reovirus variants in which the 1 attachment protein and the 2 protein that anchors 1 on the particle, are mismatched. Our studies demonstrate that a 1-2 mismatch produces particles with lower level of encapsidated 1, consequently decreasing virus attachment and infectivity. The mismatch between 1 and 2 also altered the capacity of the viral capsid to undergo conformational changes required for cell entry. These studies reveal new functions of reovirus capsid proteins, and illuminate both predictable and novel implications of reassortment.

  • Surfactin Inhibits Membrane Fusion during Invasion of Epithelial Cells by Enveloped Viruses [PublishAheadOfPrint]

  • Because membrane fusion is a crucial step in the process by which enveloped viruses invade host cells, membrane fusion inhibitors can be effective drugs against enveloped viruses. We found that surfactin from Bacillus subtilis can suppress the proliferation of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in epithelial cells at a relatively low concentration range (15-50 mmu;g/ml), without cytotoxicity or viral membrane disruption. Membrane fusion inhibition experiments demonstrate that surfactin treatment significantly reduces the rate at which virus fuses to the cell membrane. Thermodynamic experiments show that the incorporation of small amounts of surfactin hinders the formation of negative curvature by lamellar phase lipids, suggesting that surfactin acts a membrane fusion inhibitor. A fluorescent lipopeptide similar to surfactin was synthesized, and its ability to insert into the viral membrane was confirmed by spectroscopy. In vivo experiments have shown that oral administration of surfactin in piglets protects against PEDV infection. In conclusion, our study indicates that surfactin is a membrane fusion inhibitor with activity against enveloped viruses. As the first reported naturally occurring wedge lipid membrane fusion inhibitor, surfactin is likely to be a prototype for the development of a broad range of novel anti-viral drugs.

    Author summary Membrane fusion inhibitors are a rapidly emerging class of antiviral drugs that inhibit the infection process of enveloped viruses. They can be classified as fusion protein-targeting or membrane lipid-targeting, depending on the viral components targeted. Lipid-targeted membrane fusion inhibitors have a broader antiviral spectrum and are less likely to select for drug-resistant mutations. Here, we show that surfactin is a membrane fusion inhibitor and has a strong anti-viral effect. The insertion of surfactin into the viral envelope lipids reduces the probability of viral fusion. And demonstrate that oral administration surfactin protects piglets from PEDV infection. Surfactin is the first naturally occurring wedge lipid membrane fusion inhibitor that has been identified, and may be effective against many viruses beyond the scope of this study. Understanding its mechanism of action provides a foundation for the development of novel anti-viral agents.

  • In situ structures of polymerase complex and RNA genome show how aquareovirus transcription machineries respond to uncoating [PublishAheadOfPrint]

  • Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The in situ structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus, cytoplasmic polyhedrosis virus (CPV), but not for multi-layered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how RNA genome and TEC respond to priming in reoviruses is unknown. Here, we have determined the near-atomic resolution asymmetric structure of ARV at the primed state by cryo electron microscopy (cryoEM), revealing the in situ structures of 11 TECs inside each capsid, and their interactions with the 11 surrounding dsRNA genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. RdRp VP2 and NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states shows translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel is open in both states, suggesting that channel-blocking is not a regulating mechanism between these states in ARV. Instead, NTPase's C-terminal domain appears to regulate RNA translocation between quiescent and primed states. Taken together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining a similar mechanism to co-assemble their genome segments, TEC, and capsid proteins into infectious virions.

    IMPORTANCE Viruses in the family Reoviridae are characterized by their ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genome with transcriptional enzymes into a multi-layered virion and for priming such a virion for transcription are poorly understood. By cryoEM and novel asymmetric reconstruction, we have determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase, the former of which has a newly discovered domain that translocates 5' end of plus-sense RNA in segmented dsRNA genome from NTPase to polymerase when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.

  • Potent inhibition of hepatitis E virus release by a cyclic peptide inhibitor of the interaction between viral open reading frame 3 protein and host tumor susceptibility gene 101 [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) generally causes self-limiting acute viral hepatitis in normal individuals. It causes a more severe disease in immunocompromised persons and pregnant women. Due to the lack of an efficient cell culture system or animal model, life cycle of the virus is understudied, few antiviral targets are known and very few antiviral candidates have been identified against HEV infection. Inhibition of virus release is one possible antiviral development strategy, which limits spread of the virus. Earlier studies have demonstrated the essential role of the interaction between the PSAP motif of the viral open reading frame 3 protein (ORF3-PSAP) and the UEV domain of host TSG101 protein (UEV-TSG101) in mediating release of the genotype-3 HEV. Cyclic peptide (CP) inhibitors of the interaction between the Human Immunodeficiency virus (HIV) gag-PTAP motif and the UEV-TSG101 are known to block the release of HIV. Using molecular dynamic simulation, we observed that both gag-PTAP and ORF3-PSAP motifs bind to the same site in the UEV-TSG101 by hydrogen bonding. HIV release inhibitory CPs also displayed binding to the same site in the UEV-TSG101, indicating that they may compete with the ORF3-PSAP or gag-PTAP for binding to the UEV-TSG101. Two independent assays confirmed the ability of a cyclic peptide (CP11) to inhibit the ORF3-TSG101 interaction. CP11 treatment also reduced the release of both genotype-1 and genotype-3 HEV by approximately 90%, with an IC50 of 2mmu;M. Thus, CP11 appears to be an attractive candidate for further validation of its anti-HEV properties.

    Importance

    There is no specific therapy against the hepatitis E virus (HEV) induced hepatic and non-hepatic health problems. Prevention of release of the progeny viruses from the infected cells is an attractive strategy to limit spread of the virus. Interaction between the viral open reading frame 3 and the host tumor susceptibility gene 101 proteins have been shown to be essential for release of the genotype-3 HEV from the infected cells. In this study, we have identified a cyclic peptide inhibitor of the above interaction and demonstrate the efficiency of the inhibitor in preventing virus release from the infected cells. Thus, our findings uncover the possibility of developing a specific antiviral agent against the HEV by blocking its release from the infected cells.

  • PORCINE ADENOVIRUS-3 E3 ENCODES A STRUCTURAL PROTEIN ESSENTIAL FOR CAPSID STABILITY AND PRODUCTION OF INFECTIOUS PROGENY VIRIONS [PublishAheadOfPrint]

  • Adenovirus E3 region encodes proteins, which are not essential for viral replication in vitro. Porcine adenovirus-3 (PAdV-3) E3 region encodes three proteins including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K is a structural protein as it is incorporated in CsCl purified virions. The 13.7K appears essential for PAdV-3 replication as mutant PAV13.73A expressing mutated 13.7K could only be isolated in VIDO AS2 cell expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared no effect on virus assembly and production of mature virions. Further analysis of CsCl purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted broken capsids suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that PAdV-3 E3 encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.

    IMPORTANCE Although E3 region encoded proteins are involved in the modulation of leukocyte functions (Arnberg N. 2013. PNAS 110:19976-19977) and inducing lytic infection of lymphocytes (Murali VK, Ornelles DA, Gooding LR, Wilms HT, Huang W, Tollefson AE, Wold WS, Garnett-Benson C. 2014.. J virol. 88:903-912), none of the E3 proteins appear to be the component of virion capsid or required for replication of adenovirus. Here, we demonstrate that 13.7K protein encoded by E3 region of porcine adenovirus-3 is component of progeny virion capsids and appears essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that adenovirus E3 encoded protein is an essential structural protein.

  • A mutation in UL24 abolishes expression of the newly identified UL24.5 protein of herpes simplex virus 1, and leads to an increase in pathogenicity in mice [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) infects the host via epithelia and establishes latency in sensory neurons. ul24 is conserved throughout the Herpesviridae family, and the UL24 protein is important for efficient viral replication and pathogenesis. Multiple transcripts are expressed from the ul24 gene. The presence of a transcription initiation site inside the open reading frame of ul24 (ORF UL24) and an ATG start codon in the same open reading frame led us to suspect that another protein was expressed from the ul24 locus. To test our hypothesis, we constructed a recombinant virus that expresses a hemagglutin tag at the C-terminus of UL24. Western blot analysis revealed the expression of an 18 kDa protein that is not a degradation product of the full-length UL24, which we refer to as UL24.5. Ectopically expressed UL24.5 did not induce the dispersal of nucleolar proteins as seen for UL24. In order to characterize the role of UL24.5, we constructed a mutant virus encoding a substitution of the predicted initiation methionine to a valine. This substitution eliminated the expression of the 18 kDa polypeptide. Unlike the UL24-null mutant (UL24X), which exhibits reduced viral yields, the UL24.5-null mutant exhibited the same replication phenotype in cell culture as the parental strain. However, in a murine ocular infection model, we observed an increase in the incidence of neurological disorders with the UL24.5 mutant. Alignment of amino acid sequences for various herpesviruses revealed that the initiation site of UL24.5 is conserved among HSV-1 strains and is present in many herpesviruses.

    Importance

    We discovered a new HSV-1 protein, UL24.5, which corresponds to the C-terminal portion of UL24. In contrast to the replication defects observed with HSV-1 strains that do not express full length UL24, the absence of UL24.5 did not affect viral replication in cell culture. Moreover, in mice, the absence of UL24.5 did not affect viral titers in epithelia or trigeminal ganglia during acute infection; however, it was associated with a prolonged persistence of signs of inflammation. Strikingly, the absence of UL24.5 also led to an increase in the incidence of severe neurological impairment compared to results for wild-type control viruses. This increase in pathogenecity is in stark contrast to the reduction in clinical signs associated with the absence of full length UL24. Bioinformatic analyses suggest that UL24.5 is conserved among all human aalpha;-herpesviruses and in some non-human aalpha;-herpesviruses. Thus, we have identified UL24.5 as a new HSV-1 determinant of pathogenesis.

  • Role of small hydrophobic (SH) protein of J paramyxovirus (JPV) in virulence [PublishAheadOfPrint]

  • J paramyxovirus (JPV) was first isolated from moribund mice with hemorrhagic lung lesions in Australia in 1972. It is a paramyxovirus classified under the newly proposed genus, Jeilongvirus. JPV has a genome of 18,954 nucleotides, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. JPV causes little cytopathic effect (CPE) in tissue culture cells but severe disease in mice. The small hydrophobic (SH) protein is an integral membrane protein encoded by many paramyxoviruses such as mumps virus (MuV) and respiratory syncytial virus (RSV). However, the function of SH has not been defined in a suitable animal model. In this work, the functions of SH of JPV, MuV and RSV have been examined by generating recombinant JPV lacking the SH protein (rJPV-SH) or replacing SH of JPV with MuV SH (rJPV-MuVSH) or RSV SH (rJPV-RSVSH). rJPV-SH, rJPV-MuVSH, and rJPV-RSVSH were viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor-alpha (TNF-aalpha;) was produced during rJPV-SH infection, confirming the role of SH in inhibiting TNF-aalpha; production. rJPV-SH induced more apoptosis in tissue culture cells than rJPV, rJPV-MuVSH, and rJPV-RSVSH, suggesting that SH plays a role in blocking apoptosis. Furthermore, rJPV-SH was attenuated in mice compared to rJPV, rJPV-MuVSH, and rJPV-RSVSH, indicating that the SH protein plays an essential role in virulence. The results indicate that the functions of MuV SH and RSV SH are similar to that of JPV SH even though they have no sequence homology.

    IMPORTANCE Paramyxoviruses are associated with many devastating diseases in animals and humans. J paramyxovirus (JPV) was isolated from moribund mice in Australia in 1972. Newly isolated viruses such as Beilong virus (BeiPV) and Tailam virus (TlmPV) have similar genome structure of JPV. A new paramyxovirus genus, Jeilongvirus, which contains JPV, BeiPV, and TlmPV, has been proposed. Small hydrophobic (SH) protein is present in many paramyxoviruses. Our present study investigates the role of SH protein of JPV in pathogenesis in its natural host. Understanding the pathogenic mechanism of Jeilongvirus is important to control and prevent potential diseases that may emerge from this group of viruses.

  • USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1 [PublishAheadOfPrint]

  • The host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase (dNTPase). Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase (CDK) inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+ T-cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by down-regulating p21 expression and up-regulating intracellular dNTP levels. p21 down-regulation by USP18 was associated with inactive form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.

    IMPORTANCE

    Macrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1 mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The CDK inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 down-regulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

  • HIV-1 Engages a Dynein-Dynactin-BICD2 Complex for Infection and Transport to the Nucleus [PublishAheadOfPrint]

  • HIV-1 infection depends on efficient intracytoplasmic transport of the incoming viral core to the target cell nucleus. Evidence suggests that this movement is facilitated by the microtubule motor dynein, a large multi-protein complex that interacts with dynactin and cargo-specific adaptor proteins for retrograde movement via microtubules. Dynein adaptor proteins are necessary for activating dynein movement and for linking specific cargoes to dynein. We hypothesized that HIV-1 engages the dynein motor complex via an adaptor for intracellular transport. Here, we show that siRNA depletion of the dynein heavy chain, components of the dynactin complex, and the dynein adaptor BICD2, reduced cell permissiveness to HIV-1 infection. Cell depletion of dynein heavy chain and BICD2 resulted in impaired HIV-1 DNA accumulation in the nucleus and decreased retrograde movement of the virus. Biochemical studies revealed that dynein components and BICD2 associate with capsid-like assemblies of the HIV-1 CA protein in cell extracts and that purified recombinant BICD2 binds to CA assemblies in vitro. Association of dynein with CA assemblies was reduced upon immunodepletion of BICD2 from cell extracts. We conclude that BICD2 is a capsid-associated dynein adaptor utilized by HIV-1 for transport to the nucleus.

    Importance

    During HIV-1 infection, the virus must travel across the cytoplasm to enter the nucleus. The host cell motor protein complex dynein has been implicated in HIV-1 intracellular transport. We show that expression of the dynein heavy chain, components of the dynein-associated dynactin complex, and the dynein adaptor BICD2 in target cells are important for HIV-1 infection and nuclear entry. BICD2 interacts with the HIV-1 capsid in vitro, suggesting that it functions as a capsid-specific adaptor for HIV-1 intracellular transport. Our work identifies specific host proteins involved in microtubule-dependent HIV-1 intracellular transport and highlights the BICD2-capsid interaction as a potential target for antiviral therapy.

  • The Effect of Productive HPV16 Infection on Global Gene Expression of Cervical Epithelium [PublishAheadOfPrint]

  • Human papillomavirus (HPV) infection is the world's most common sexually transmitted infection and is responsible for most cases of cervical cancer. Previous studies of global gene expression changes induced by HPV infection have focused on the cancerous stages of infection, and therefore, not much is known about global gene expression changes at early pre-neoplastic stages of infection. We show for the first time, global gene expression changes of early stage HPV16 infection in cervical tissue using 3-dimensional organotypic raft cultures that produce high levels of progeny virions.

    cDNA microarray analysis showed that a total of 594 genes were upregulated and 651 genes were downregulated at least 1.5-fold with HPV16 infection. Gene ontology analysis showed that biological processes including cell cycle progression and DNA metabolism were upregulated, while skin development, immune response, and cell death were downregulated with HPV16 infection in cervical keratinocytes. Individual genes were selected for validation at the transcriptional and translational levels including UBC, which was central to the protein association network of immune response genes, and top downregulated genes RPTN, SERPINB4, KRT23, and KLK8. In particular, KLK8 and SERPINB4 have shown to be upregulated in cancer, which contrasts the productive replication stage.

    Organotypic raft cultures that allow full progression of the HPV life-cycle have allowed us to identify novel gene modulations and potential therapeutic targets of early stage HPV infection in cervical tissue. Additionally, our results suggest that early stage productive infection and cancerous stages of infection are distinct disease states expressing different host transcriptomes.

    Importance Persistent HPV infection is responsible for most cases of cervical cancer. Transition from precancerous to cancerous stages of HPV infection is marked by a significant reduction in virus production. Most global gene expression studies of HPV infection have focused on the cancerous stages. Therefore, little is known about global gene expression changes at precancerous stages. For the first time, we measured global gene expression changes at precancerous stages of HPV16 infection in human cervical tissue producing high levels of virus. We identified a group of genes that are typically overexpressed in cancerous stages to be significantly downregulated at the precancerous stage. Moreover, we identified significantly modulated genes that have not yet been studied in the context of HPV infection. Studying the role of these genes in HPV infection will help us understand what drives the transition from precancerous to cancerous stages, and may lead to development of new therapeutic targets.

  • Differential Mechanisms for the Involvement of Polyamines and Hypusinated eIF5A in Ebola Virus Gene Expression [PublishAheadOfPrint]

  • Polyamines and hypusinated eIF5A have been implicated in the replication of diverse viruses, however, defining their roles in supporting virus replication is still under investigation. We have previously reported that Ebola virus (EBOV) requires polyamines and hypusinated eIF5A for replication. Using a replication deficient minigenome construct, we show that gene expression, in the absence of genome replication, requires hypusinated eIF5A. Additional experiments demonstrated the block in gene expression upon hypusine depletion was post-transcriptional, as minigenome reporter mRNA transcribed by the EBOV polymerase accumulated normally in the presence of drug treatment where protein did not. When this mRNA was isolated from cells with low levels of hypusinated eIF5A and transfected into cells with normal eIF5A function, minigenome reporter protein accumulation was normal, demonstrating that the mRNA produced was functional but required hypusinated eIF5A function for translation. Our results support a mechanism in which hypusinated eIF5A is required for the translation of, but not synthesis of, EBOV transcripts. In contrast, depletion of polyamines with DFMO resulted in a strong block in the accumulation of EBOV polymerase-produced mRNA, indicating a distinct mechanism of polyamine suppression of EBOV gene expression. Supplementing with exogenous polyamines after DFMO treatment restored mRNA accumulation and luciferase activity. These data indicate cellular polyamines are required for two distinct aspects of the EBOV lifecycle. The bifunctional requirement for polyamines underscores the importance of these cellular metabolites in EBOV replication and suggests that repurposing existing inhibitors of this pathway could be an effective approach for EBOV therapeutics.

    IMPORTANCE: Ebolavirus is a genetically simple virus that has a small number of proteins. Because of this, it requires host molecules and proteins to produce new infectious virus particles. Though attention is often focused on cellular proteins required for this process, it has recently been shown that cellular metabolites such as polyamines are also necessary for EBOV replication. Here we show that polyamines such as spermine and spermidine are required for the accumulation of EBOV mRNA and that eIF5A, a molecule modified by spermidine, is required for the translation of, but not the production of, EBOV mRNAs. These findings suggest that effectively targeting this pathway could provide a biphasic block of EBOV replication.

  • Evasion Strategy of Human Cytomegalovirus to Escape Interferon-{beta}-Induced APOBEC3G Editing Activity [PublishAheadOfPrint]

  • The apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) and to a lesser degree A3F, but not A3A, gene products are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon-bbeta; (IFN-bbeta;), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-bbeta; by limiting the presence of A3G hotspots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.

    IMPORTANCE APOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study we show that particularly APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and its upregulation is mediated by IFN-bbeta;. Furthermore, we were able to demonstrate that neither A3G knock out nor its overexpression appear to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hotspots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteract mechanisms under A3G-mediated selective pressure.

  • Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice [PublishAheadOfPrint]

  • The hemagglutinin protein of H3N2 influenza viruses is the major target of neutralizing antibodies induced by infection and vaccination. However, the virus frequently escapes antibody-mediated neutralization due to mutations in the globular head domain. Five topologically distinct antigenic sites in the head domain of H3 hemagglutinin, A-E, have been previously described by mapping the binding sites of monoclonal antibodies. Yet, little is known about the contribution of each site to the immunogenicity of modern H3 hemagglutinins, as measured by hemagglutination inhibition activity that is known to correlate with protection. To investigate the hierarchy of antibody immunodominance, five llsquo;1rrsquo; recombinant influenza viruses expressing hemagglutinin of the A/Hong Kong/4801/2014 (H3N2) strain with mutations in single antigenic sites were generated. Next, the 1 viruses were used to determine the hierarchy of immunodominance by measuring the hemagglutination inhibition reactivity of mouse antisera and plasma from 18 human subjects before and after seasonal influenza vaccination in 2017/2018. In both mice and humans, mutations in antigenic site B caused the most significant decrease in hemagglutination inhibition titers compared to wildtype hemagglutinin. This study revealed that antigenic site B is immunodominant in the H3N2 influenza virus strain included in the current vaccine preparations.

    IMPORTANCE Influenza viruses rapidly evade humoral immunity through antigenic drift, making current vaccines poorly effective and antibody-mediated protection short-lived. The majority of neutralizing antibodies target five antigenic sites in the head domain of the hemagglutinin protein that are also the most sequence-variable regions. A better understanding of the contribution of each antigenic site to the overall antibody response to hemagglutinin may help in the design of improved influenza virus vaccines.

  • RNA polymerase mutations selected during experimental evolution enhance replication of a hybrid vaccinia virus with an intermediate transcription factor subunit replaced by the myxoma virus ortholog [PublishAheadOfPrint]

  • High-throughput DNA sequencing enables the study of experimental evolution in near real time. Until now, mutants with deletions of non-essential host range genes were used in experimental evolution of vaccinia virus (VACV). Here, we guided the selection of adaptive mutations that enhanced the fitness of a hybrid virus in which an essential gene had been replaced with an ortholog from another poxvirus genus. Poxviruses encode a complete system for transcription including RNA polymerase and stage-specific transcription factors. The abilities of orthologous intermediate transcription factors from other poxviruses to substitute for those of VACV, determined by transfection assays, corresponded with the degree of amino acid identity. VACV in which the A8 or A23 intermediate transcription factor subunit gene was replaced by the myxoma (MYX) virus ortholog exhibited decreased replication. During three parallel serial passages of the hybrid virus with the MYXA8 gene, plaque sizes and virus yields increased. DNA sequencing of virus populations at passage 10 revealed high frequencies of five different single nucleotide mutations in the two largest RNA polymerase subunits RPO147 and RPO132, and two different Kozak consensus sequence mutations predicted to increase translation of the MYXA8 mRNA. Surprisingly, there were no mutations within either intermediate transcription factor subunit. Based on homology with yeast RNA polymerase, the VACV mutations were predicted to be buried within the internal structure of the enzyme. By directly introducing single nucleotide substitutions into the genome of the original hybrid virus, both RNA polymerase and translation-enhancing mutations were demonstrated to increase virus replication independently.

    IMPORTANCE Previous studies demonstrated the experimental evolution of vaccinia virus (VACV) following deletion of a host range gene important for evasion of host immune defenses. We have extended experimental evolution to essential genes that cannot be deleted but could be replaced by a divergent orthologous gene from another poxvirus. Replacement of a VACV transcription factor gene with one from a distantly related poxvirus led to decreased fitness evidenced by diminished replication. Serially passaging the hybrid virus at a low multiplicity provided conditions for selection of adaptive mutations that improved replication. Notably, these included five independent mutations of the largest and second largest RNA polymerase subunits. This approach should be generally applicable for investigating adaptation to swapping of orthologous genes encoding additional essential proteins of poxviruses as well as other viruses.

  • INOSINE TRIPHOSPHATE PYROPHOSPHATASE DEPHOSPHORYLATES RIBAVIRIN TRIPHOSPHATE AND REDUCED ENZYMATIC ACTIVITY POTENTIATES MUTAGENESIS IN HEPATITIS C VIRUS [PublishAheadOfPrint]

  • Background and aims: A third of humans carry genetic variants of the inosine triphosphate pyrophosphatase (ITPase) gene (ITPA) entailing reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia, and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been forwarded to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. Methods: The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with siRNA directed against ITPA or negative control siRNA in the presence or absence of ribavirin in an HCV culture system. Results: Low ribavirin concentrations strikingly depleted intracellular guanosine triphosphate (GTP) levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions into the HCV genome with ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as inosine triphosphate (ITP), a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Conclusions: Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.

    IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular guanosine triphosphate and increased hepatitis C virus mutagenesis. In cell culture, reduced inosine triphosphate pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP is dephosphorylated in vitro by ITPase, similar to inosine triphosphate (ITP), which is a naturally occurring substrate of ITPase.

  • MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2 [PublishAheadOfPrint]

  • MicroRNAs (miRNAs) are small non-coding RNAs that are crucial post-transcriptional regulator for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA viruses infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we show that expression of cellular miRNA, miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses, H5N1, A/PR8/H1N1, and NDV and transfection with polyI:C. We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of Type-I, Type-III interferons, and interferons-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of JAK1-STAT3 pathway. Altogether, these findings highlights that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.

    IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003-2017, H5N1 HPAI has caused 453 human deaths with a high mortality rate of 52.74%. This work shows that miR-324-5p suppresses the H5N1 HPAI viral replication by directly targeting viral genome (thereby inhibits viral gene expression) and cellular CUEDC2 gene, the negative regulator of interferon pathway (thereby enhance anti-viral genes). Our study enhance the knowledge of role of microRNAs in cellular response to viral infection. Also, the study provides help in understanding how the host cells utilizes small RNAs in controlling the viral burden.

  • The Herpes Simplex Virus 1 {gamma}134.5 Protein Inhibits STING Activation That Restricts Viral Replication [PublishAheadOfPrint]

  • The 134.5 gene of herpes simplex virus 1 (HSV-1) encodes a virulence factor that promotes viral pathogenesis. Although it perturbs TANK-binding kinase 1 (TBK1) in the complex network of innate immune pathways, the underlying mechanism is obscure. Here we report that HSV-1 134.5 targets stimulator of interferon genes (STING) in the intracellular DNA recognition pathway that regulates TBK1 activation. In virus-infected cells the 134.5 protein associates with and inactivates STING, which leads to downregulation of interferon regulatory factor 3 (IRF3) and IFN responses. Importantly, HSV-1 134.5 disrupts translocation of STING from the endoplasmic reticulum to Golgi apparatus, a process necessary to prime cellular immunity. Deletion of 134.5 or its amino-terminal domain from HSV-1 abolishes the observed inhibitory activities. Consistently, an HSV mutant that lacks functional 134.5 replicates less efficiently in STING+/+ than in STING-/- mouse embryonic fibroblasts. Moreover, reconstituted expression of human STING in the STING-/- cells activates IRF3 and reduces viral growth. These results suggest that control of the DNA sensing pathway by 134.5 is advantageous to HSV infection.

    IMPORTANCE Viral inhibition of innate immunity contributes to herpes simplex virus pathogenesis. Although this complex process involves multiple factors, the underlying events remain unclear. We demonstrate that an HSV virulence factor 134.5 precludes the activation of STING, a central adaptor in the intracellular DNA sensing pathway. Upon HSV infection, this viral protein engages with and inactivates STING. Consequently, it compromises host immunity and facilitates HSV replication. These observations uncover an HSV mechanism that is likely to mediate viral virulence.

  • Multi-Antigenic Modified Vaccinia Ankara Vaccine Vectors to Elicit Potent Humoral and Cellular Immune Reponses Against Human Cytomegalovirus in Mice [PublishAheadOfPrint]

  • As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet, an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in the protection against HCMV infection and disease. Here, we describe a vaccine approach based on the well-characterized Modified Vaccinia Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show in mice that multi-antigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multi-antigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate in vitro neutralization of different HCMV strains and antibody-dependent cellular cytotoxicity. These results support the use of MVA to develop a multi-antigenic vaccine candidate for controlling HCMV infection and disease in different target populations such as pregnant women and transplant recipients.

    IMPORTANCE The development of a human cytomegalovirus (HCMV) vaccine to prevent congenital disease and transplantation-related complications is an unmet medical need. While many HCMV vaccine candidates have been developed, partial success to prevent or control HCMV infection in women of childbearing age and transplant recipients has been observed with an approach based on envelope glycoprotein B (gB). We introduce a novel vaccine strategy based on the clinically-deployable Modified Vaccinia Ankara (MVA) vaccine vector to elicit potent humoral and cellular immune responses by multiple immunodominant HCMV antigens, including gB, phosphoprotein 65, and all five subunits of the pentamer complex. These findings could contribute to develop a multi-antigenic vaccine strategy that may afford improved protection against HCMV infection and disease than a vaccine approach employing solely gB.

  • The HIV integrase inhibitor raltegravir inhibits felid alphaherpesvirus 1 (FeHV-1) replication by targeting both DNA replication and late gene expression. [PublishAheadOfPrint]

  • Alphaherpesvirus-associated ocular infections in humans, caused by human alphaherpesvirus 1 (HHV-1), remain challenging to treat due to the frequency of drug application required and the potential for the selection of drug resistant viruses. Repurposing on-the-market drugs is a viable strategy to accelerate the pace of drug development. It has been reported that the human immunodeficiency virus (HIV) integrase inhibitor raltegravir inhibits HHV-1 replication by targeting the DNA polymerase accessory factor and limits terminase-mediated genome cleavage of the human betaherpesvirus 5 (HHV-5). We have previously shown, both in vitro and in vivo, that raltegravir can also inhibit the replication of felid alphaherpesvirus 1 (FeHV-1), a common ocular pathogen of cats with a similar pathogenesis to HHV-1 ocular disease. In contrast to what was reported for HHV-1, we were unable to select for a raltegravir-resistant FeHV-1 in order to define any basis for drug action. A candidate-based approach to explore the mode-of-action of raltegravir against FeHV-1 showed that raltegravir did not impact FeHV-1 terminase function, as described for HHV-5. Instead, raltegravir inhibited DNA replication, similar to HHV-1, but by targeting the initiation of viral DNA replication rather than elongation. In addition, we found that raltegravir specifically repressed late gene expression independent of DNA replication, and both activities are consistent with inhibition of ICP8. Taken together, these results suggest that raltegravir could be a valuable therapeutic agent against herpesviruses.

    Importance: The rise of drug-resistant herpesviruses is a long-standing concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that this drug could prove a viable strategy for the treatment of herpesvirus infections.

  • Genetic evidence supports sporadic and independent introductions of subtype H5 low pathogenic avian influenza A viruses from wild birds to domestic poultry in North America [PublishAheadOfPrint]

  • Wild birdnndash;origin influenza A viruses (IAVs or avian influenza) have led to sporadic outbreaks among domestic poultry in the United States (US) and Canada, resulting in economic losses through the implementation of costly containment practices and destruction of birds. We used evolutionary analyses of virus sequence data to determine that 78 H5 low pathogenic avian influenza viruses (LPAIVs) isolated from domestic poultry in the US and Canada during 2001nndash;2017 resulted from 18 independent virus introductions from wild birds. Within the wild bird reservoir, the hemagglutinin gene segments of H5 LPAIVs exist primarily as two co-circulating genetic sublineages, and our findings suggest the H5 gene segments flow within each migratory bird flyway and among adjacent flyways, with limited exchange between the non-adjacent Atlantic and Pacific Flyways. Phylogeographic analyses provided evidence that IAVs from dabbling ducks and swans/geese contributed to emergence of viruses among domestic poultry. H5 LPAIVs isolated from commercial farm poultry (i.e. turkey) were descended from a single introduction typically remain a single genotype, whereas those from live bird markets sometimes led to multiple genotypes, reflecting the potential for reassortment with other IAVs circulating within live bird markets. H5 LPAIV introduced from wild birds to domestic poultry represent economic threats to the U.S. poultry industry, and our data suggest that such introductions have been sporadic, controlled effectively through production monitoring and a stamping-out policy, and are, therefore, unlikely to result in sustained detections in commercial poultry operations.

    IMPORTANCE Integration of viral genome sequencing into influenza surveillance for wild birds and domestic poultry can elucidate evolutionary pathways of economically costly poultry pathogens. Evolutionary analyses of H5 LPAIVs detected in domestic poultry in US and Canada during 2001nndash;2017 suggest that these viruses originated from repeated introductions of IAVs from wild birds, followed by various degrees of reassortment. Reassortment was observed where biosecurity was low and there were opportunities for more than one virus to circulate existed (e.g. congregations of birds from different premises such as live bird markets). None of the H5 lineages identified were maintained long term in domestic poultry, suggesting that management strategies have been effective in minimizing the impacts of virus introductions on US poultry production.

  • The human cytomegalovirus ER resident glycoprotein UL148 activates the unfolded protein response. [PublishAheadOfPrint]

  • Eukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol requiring enzyme-1 (IRE1), as indicated by splicing of Xbp1 mRNA, and the PKR-like ER kinase (PERK), as indicated by phosphorylation of eIF2aalpha; and accumulation of ATF4. During wild-type HCMV infection, increases in Xbp-1 splicing, eIF2aalpha; phosphorylation and accumulation of ATF4 accompany UL148 expression. UL148-null infections, however, show reduced levels of these UPR indicators, and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. siRNA depletion of PERK dampened the extent of eIF2aalpha; phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2aalpha; kinases. A virus disrupted for UL148 showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV infected cell.

    IMPORTANCE The unfolded protein response (UPR) is an ancient cellular response to ER stress of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shut-off, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER resident glycoprotein UL148 contributes substantially to the UPR during infection, and moreover is sufficient to activate the UPR in non-infected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell mediated immunity.

  • Sera from individuals with narrowly focused influenza virus antibodies rapidly select viral escape mutations in ovo [PublishAheadOfPrint]

  • Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo. We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals that produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.

    IMPORTANCE Influenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.

  • Bovine herpesvirus 1 entry by a low pH endosomal pathway [PublishAheadOfPrint]

  • Bovine herpesvirus (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (t1/2 of ~ 5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times post-infection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane, and necessary for successful infection of the target cell.

    IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they limit disease severity and spread, but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses such as HSV-1, which has been more widely addressed.

  • Herpes simplex virus type 1 infection promotes the growth of a subpopulation of tumor cells in 3D uveal melanoma cultures [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1)-mediated oncolytic therapy is an emerging cancer treatment modality with potential effectiveness against a variety of malignancies. To better understand the interaction of HSV-1 with neoplastic cells, we inoculated three-dimensional (3D) cultures of human uveal melanoma cells with HSV-1. 3D melanoma cultures were established by placing tumor cells on the surface of a Matrigel matrix which was followed by the growth of tumor cells on the matrix surface and invasion of the Matrigel matrix by some tumor cells to form multicellular tumor spheroids within the matrix. When established 3D melanoma cultures were inoculated with HSV-1 by placing virus on the surface of cultures, virus infection caused extensive death of melanoma cells growing on the surface of the 3D matrix and significantly decreased the number of tumor cell spheroids within the matrix. However, HSV-1 infection did not lead to a complete destruction of tumor cells in the 3D cultures during a 17-day observation period and surprisingly, HSV-1 infection promoted the growth of some melanoma cells within the matrix as determined by a significantly increased size of residual viable multicellular tumor spheroids in virus-inoculated 3D cultures at 17 days after virus inoculation. Acyclovir treatment inhibited HSV-1-induced tumor cell killing, but did not block virus infection-induced increase in spheroid size. These findings suggest that although HSV-1 oncolytic virotherapy may cause extensive tumor cell killing it may also be associated with unintended promotion of the growth of some tumor cells.

    IMPORTANCE Cancer cells are exposed to HSV-1 during oncolytic virotherapy with an intension of tumor cell killing. Our observations reported here suggest that potential dangers of HSV-1 oncolytic therapy include promotion of growth of some tumor cells. Furthermore, our findings raise the possibility that HSV-1 infection of neoplastic cells during natural infections or vaccinations may promote the growth of tumors. Our study indicates that HSV-1 infection of 3D tumor cell cultures provides an experimental platform in which mechanisms of HSV-1-mediated promotion of tumor cell growth can be effectively studied.

  • FUNCTIONAL EVOLUTION OF THE 2009 PANDEMIC H1N1 INFLUENZA NS1 AND PA IN HUMANS [PublishAheadOfPrint]

  • In 2009, an H1N1 influenza A virus (IAV, pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding for proteins inhibiting general gene expression (non-structural protein 1, NS1; and PA-X) from circulating seasonally viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E and N205S), as previously described (1), and amino acid changes in the PA-X protein (V100I, N204S, R221Q and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V and R362K). A recombinant pH1N1 virus containing PA, PA-X and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of the pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness.

    IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009 and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and nowadays were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo. Importantly, these results suggest that a balance in the ability of NS1 and PA-X to induce host shutoff is beneficial for IAVs.

  • Kaposi sarcoma-associated herpesvirus latency locus renders B cells hyperresponsive to secondary infections [PublishAheadOfPrint]

  • Kaposi Sarcoma-associated herpesvirus (KSHV) KSHV induces B cell hyperplasia and neoplasia such as multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). To explore KSHV-induced B cell reprogramming in vivo, we expressed the KSHV latency locus, inclusive of all viral miRNAs, in B cells of transgenic mice in the absence of the inhibitory FcRIIB receptor. The BALB/c strain was chosen as this is the preferred model to study B cell differentiation. The mice developed hyperglobulinemia, plasmacytosis, and B lymphoid hyperplasia. This phenotype was ameliorated by everolimus, which is a rapamycin-derivative approved for the treatment of mantle cell lymphoma. KSHV-latency mice exhibited hyperresponsiveness to the T-dependent (TD) antigen mimic aalpha;-CD40 and increased incidence of pristane-induced inflammation. Lastly, the adaptive immunity against a secondary infection with Zika virus (ZIKV) was markedly enhanced. These phenotypes are consistent with KSHV lowering the activation threshold of latently-infected B cells, which may be beneficial in endemic areas, where KSHV is acquired in childhood and where infections are common.

    IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) establishes latency in B cells and is stringently linked to primary effusion lymphoma (PEL) and the pre-malignant B cell hyperplasia multicentric Castleman's disease (MCD). To investigate potential genetic background effects, we expressed the KSHV miRNAs in BALB/c transgenic mice. BALB/c are the preferred strain for B cell hybridoma development because of their propensity to develop predictable B cell responses to antigen. The BALB/c-latency mice exhibited a higher incidence of B cell hyperplasia as well as sustained hyperglobulinemia. The development of neutralizing antibodies against ZIKV was augmented in BALB/c-latency mice. Hyperglobulinemia was dampened by everolimus, a derivative of rapamycin, suggesting a role for mTOR inhibitors in managing immune activation, which is hallmark of KSHV infection as well as HIV infection.

  • Testicular degeneration and infertility following arbovirus infection [PublishAheadOfPrint]

  • Arboviruses can cause a variety of clinical signs including febrile illness, arthritis, encephalitis and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1IT2006) or low (BTV-1IT2013) virulence strain of BTV. Rams infected with the low virulent BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type-I IFN response, reduction of testosterone biosynthesis by Leydig cells, and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily.

    Importance

    During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type-I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.

  • Phosphatase Cdc25A negatively regulates antiviral immune response by inhibiting TBK1 activity [PublishAheadOfPrint]

  • Phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of IFN-bbeta; induced by Sendai virus and poly(I:C), while knockdown of Cdc25A enhances the transcription of IFN-bbeta; stimulated by RNA virus infection. The inhibitory effect of Cdc25A on antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-bbeta;. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A up-regulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits antiviral immune response by reducing the active form of TBK1. Using HSV-1 infection, an IFN-bbeta; reporter assay and RT-qPCR, we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-bbeta;. Using a VSV infection assay, we confirmed that Cdc25A can repress RLR-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates antiviral immune response through inhibiting TBK1 activity.

    IMPORTANCE RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism of balancing the RLR signaling pathway in host cells is not well understood. We found that phosphatase Cdc25A negatively regulates RNA virus-induced innate immune response. Our studies indicated that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-bbeta; transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-bbeta;. Taken together, our findings uncover a novel function and mechanism of Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity, demonstrating its role in maintaining host cell homeostasis following viral infection.

  • SOSIP changes affect human immunodeficiency virus (HIV-1) envelope glycoprotein conformation and CD4 engagement [PublishAheadOfPrint]

  • The entry of human immunodeficiency virus into the host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more open downstream State-2 and State-3 conformations. Changes in "restraining" amino acid residues such as leucine 193 and isoleucine 423 destabilize State-1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State-1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes and well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented herein suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.

    IMPORTANCE

    The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on Env conformation, moving Env away from the native pre-triggered Env conformation. Our studies underscore the need to acquire structural information on the pre-triggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.

  • Mutant cellular AP-1 proteins promote expression of a subset of Epstein-Barr virus late genes in the absence of lytic viral DNA replication [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here we compare expression of 67 viral genes induced by ZEBRA versus induced AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes, when compared to ZEBRA, is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune-modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune-modulator.

    IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provides proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

  • The S2 subunit of infectious bronchitis virus Beaudette is a determinant of cellular tropism [PublishAheadOfPrint]

  • The spike glycoprotein (S) of the avian gammacoronavirus, infectious bronchitis virus (IBV), is comprised of two subunits, S1 and S2, and has a role in virulence in vivo as well as being responsible for cellular tropism in vitro. We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells we have generated a series of recombinant IBVs expressing chimaeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability for IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just three amino acids surrounding the S2' cleavage site. Conversely, swapping the three Beaudette-associated amino acids with corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.

    IMPORTANCE Infectious Bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism have been identified in this study, which will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.

  • Emerging pro-viral roles of caspases during lytic replication of gammaherpesviruses [PublishAheadOfPrint]

  • Due to their roles in the regulation of programmed cell death and inflammation, the cellular caspase proteases are considered anti-viral factors. However, recent studies have revealed examples of pro-viral functions for caspases. Here we review a growing body of literature on the role of caspases in promoting the replication of human gammaherpesviruses. We propose that gammaherpesviruses have evolved ways to redirect these enzymes and to use their activation to support viral replication and immune evasion.

  • Oral prion neuroinvasion occurs independently of PrPC expression in the gut epithelium [PublishAheadOfPrint]

  • The early replication of certain prion strains within the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally-acquired prions utilise specialised gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in the Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.

    IMPORTANCE The accumulation of orally-acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within the Peyer's patches. Some gastrointestinal pathogens utilize molecules such as the cellular prion protein, PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally-acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility.

  • The RNA-binding protein of a double-stranded RNA virus acts like a scaffold protein [PublishAheadOfPrint]

  • Infectious bursal disease virus (IBDV), a non-enveloped, double-stranded (ds)RNA virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed dsDNA viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy, cryo-electron tomography and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ~40 AAring;-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded (ss)RNA viruses.

    IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report cryo-EM and cryo-electron tomography structures and atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome-bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendent of ancestral viruses, and comprises different features of current viral lineages.

  • Sendai Virus V Protein Inhibits the Secretion of Interleukin-1{beta} by Preventing NLRP3 Inflammasome Assembly [PublishAheadOfPrint]

  • Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1bbeta; (IL-1bbeta;) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1bbeta; maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus (SeV V(-)) induced markedly greater amounts of IL-1bbeta; than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1bbeta; secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1bbeta; activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation was observed between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1bbeta; secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1bbeta; activation.

    IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also identify novel therapeutic targets for paramyxovirus infection and related diseases.

  • Growth-adaptive mutations in the Ebola Makona glycoprotein alter different steps in the virus entry pathway. [PublishAheadOfPrint]

  • The Zaire ebolavirus (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped VSV, two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV VLPs. The gain of entry function mapped to two mechanistic phenotypes. Mutations in HR1 decreased the requirement for cathepsin-B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin-B dependence. Several mutations in the fusion loop were substitutions to residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure.

    IMPORTANCE Zaire ebolavirus (EBOV) is the causative agent of the highly lethal Ebola Virus Disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking however promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recent years, distinguishing between tissue-culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here we utilize rVSV pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

  • Hepatitis B virus deregulates cell cycle to promote viral replication and a premalignant phenotype [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection is a major health problem worldwide and chronically infected individuals are at high risk of developing cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms whereby HBV causes HCC are largely unknown. By using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expressions and signaling pathways of infected hepatocytes, and whether these effects are relevant to productive HBV infection and HBV-associated HCC. Using a human growth factor antibody array, we first showed that HBV infection induced a distinct profile of growth factor production by PHHs, marked particularly by significantly lower levels of transforming growth factor (TGF)-bbeta; family of proteins in the supernatant. Transcriptome profiling next revealed multiple changes in cell proliferation and cell cycle control pathways in response to HBV infection. A human cell cycle PCR array validated deregulation of more than 20 gene associated with cell cycle in HBV-infected PHHs. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase as compared to the predominantly G0/G1 phase of cultured PHHs. HBV proviral host factors, such as PPARA, RXRA and CEBPB, were up-regulated upon HBV infection and particularly enriched in cells at the G2/M phase. Together, these results support that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive HBV infection. By perturbing cell cycle regulation of infected cells, HBV may coincidently induce a premalignant phenotype that predispose infected hepatocytes to subsequent malignant transformation.

    IMPORTANCE Hepatitis B virus (HBV) infection is a major health problem with high risk of developing hepatocellular carcinoma (HCC). By using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expressions, and whether these effects are relevant to HBV-associated HCC. HBV induced a distinct profile of growth factor production, marked particularly by significantly lower levels of transforming growth factor (TGF)-bbeta; family of proteins. Transcriptome profiling revealed multiple changes in cell proliferation and cell cycle control pathways. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase. HBV proviral host factors were up-regulated upon infection and particularly enriched in cells at the G2/M phase. Together, these results support that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive infection. This may coincidently induce a premalignant phenotype that predispose infected hepatocytes to subsequent malignant transformation.

  • Analysis of complement-mediated lysis of SIV and SIV-infected cells reveals sex differences in vaccine-induced immune responses in Rhesus macaques [PublishAheadOfPrint]

  • An effective HIV vaccine has yet to be developed and defining immune correlates of protection against HIV infection is of paramount importance to inform future vaccine design. The complement system is a component of innate immunity that can directly lyse pathogens and shape adaptive immunity. To determine if complement lysis of SIV and/or SIV-infected cells represents a protective immune correlate against SIV infection, sera from previously vaccinated and challenged Rhesus macaques were analyzed for the induction of antibody-dependent complement-mediated lysis (ADCML). Importantly, the vaccine regimen, consisting of a replication-competent adenovirus type 5 host-range mutant SIV recombinant prime followed by a monomeric gp120 or oligomeric gp140 boost, resulted in overall delayed SIV acquisition only in females. Here, sera from all vaccinated animals induced ADCML of SIV and SIV-infected cells efficiently, regardless of sex. A modest correlation of SIV lysis with reduced infection rate in males but not females, together with reduced peak viremia in all animals boosted with gp140 suggested a potential for influencing protective efficacy. Gag-specific IgG, and gp120-specific IgG and IgM, correlated with SIV lysis in females while Env-specific IgM correlated with SIV-infected cell lysis in males indicating sex differences in vaccine-induced antibody characteristics and function. In fact, gp120-/gp140-specific antibody functional correlates between ADCC, ADCP, and ADCML as well as gp120-specific IgG glycan profiles and corresponding ADCML correlations varied depending on the sex of the vaccinees. Overall, these data suggest that sex influences vaccine-induced antibody function which should be considered in design of globally-effective HIV vaccines in the future.

    Importance

    An HIV vaccine would thwart the spread of HIV infection and save millions of lives. Unfortunately, immune responses conferring universal protection from HIV infection are poorly defined. The innate immune system, including the complement system, is an evolutionarily conserved, basic means of protection from infection. Complement can prevent infection by directly lysing incoming pathogens. We found that vaccination against SIV in Rhesus macaques induces antibodies that are capable of directing complement lysis of SIV and SIV-infected cells in both sexes. We also found sex differences in vaccine-induced antibody species and their functions. Overall, our data suggest that sex affects vaccine-induced antibody characteristics and function and that males and females might require different immune responses to protect against HIV infection. This information could be used to generate highly effective HIV vaccines for both sexes in the future.

  • Enhancer control of miR-155 expression in Epstein-Barr virus infected B cells [PublishAheadOfPrint]

  • The oncogenic microRNA miR-155 is the most frequently upregulated miRNA in Epstein-Barr virus (EBV)-positive B cell malignancies and is upregulated in other non-viral lymphomas. Both the EBV nuclear antigen 2 (EBNA2), and B cell transcription factor, interferon regulatory factor 4 (IRF4) are known to activate transcription of the host cell gene from which miR-155 is processed (miR-155HG, BIC). EBNA2 also activates IRF4 transcription indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation of IRF4 and miR-155HG by EBNA2 however has not been defined. We demonstrate that EBNA2 can activate IRF4 and miR-155HG expression through specific upstream enhancers that are dependent on the Notch signaling transcription factor RBPJ, a known binding partner of EBNA2. We demonstrate that in addition to activation of the miR-155HG promoter, IRF4 can also activate miR-155HG via the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsive miR-155HG enhancer located 60 kb upstream of miR-155HG led to reduced miR155HG expression in EBV-infected cells. Our data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-miR-155 expression network and play a key role in the maintenance of miR-155 expression in EBV-infected B cells. These findings provide important insights that will improve our understanding of miR-155 control in B cell malignancies.

    IMPORTANCE MicroRNA-155 (miR-155) is expressed at high level in many human cancers particularly lymphomas. Epstein-Barr virus (EBV) infects human B cells and drives the development of numerous lymphomas. Two EBV-encoded genes (LMP1 and EBNA2) upregulate miR-155 expression and miR-155 expression is required for the growth of EBV-infected B cells. We show that the EBV transcription factor EBNA2 upregulates miR-155 expression by activating an enhancer upstream from the miR-155 host gene (miR-155HG) from which miR-155 is derived. We show that EBNA2 also indirectly activates miR-155 expression through enhancer-mediated activation of IRF4. IRF4 then activates both the miR-155HG promoter and the upstream enhancer, independently of EBNA2. Gene editing to remove the miR-155HG enhancer leads to a reduction in miR-155HG expression. We therefore identify enhancer-mediated activation of miR-155HG as a critical step in promoting B cell growth and a likely contributor to lymphoma development.

  • Antisense-Derived HIV-1 Cryptic Epitopes Are Not Major Drivers of Viral Evolution during the Acute Phase of Infection [PublishAheadOfPrint]

  • While prior studies have demonstrated that CD8 T cell responses to cryptic epitopes (CE) are readily detectable during HIV-1 infection, their ability to drive escape mutations following acute infection is unknown. We predicted 66 CE in a Zambian acute infection cohort based on escape mutations occurring within or near the putatively predicted HLA-I restricted epitope. The CE were evaluated for CD8 T cell responses in patients with chronic and acute HIV infection. Of the 66 predicted CE, 10 were recognized in 8/32 and 4/11 patients with chronic, and acute infection respectively. The immunogenic CE were all derived from a single antisense reading frame within pol. However, when these CE were tested using longitudinal study samples, CE specific T cell responses were detected but did not consistently select for viral escapes. Thus, while we demonstrated that CE are immunogenic in acute infection, the immune responses to CE are not major drivers of viral escape in the initial stages of HIV infection. This latter finding may be due to either the subdominant nature of CE-specific responses, the low antigen sensitivity, and magnitude of CE responses during acute infections.

    IMPORTANCE Although prior studies demonstrated that cryptic epitopes of HIV-1 induce CD8 T cell responses, evidence supporting that targeting these epitopes to drive HIV escape mutations have been substantially limited and none have addressed this question following acute infection. In this comprehensive study, we utilized longitudinal viral sequencing data obtained from three separate acute infection cohorts to predict potential cryptic epitopes based on HLA-I associated viral escape. Our data shows that cryptic epitopes are immunogenic during acute infection and many of these responses are elicited towards translation products of HIV-1 antisense reading frames. However, despite cryptic epitope targeting, our study did not find any evidence of early CD8 mediated immune escape. Nevertheless, improving cryptic epitope specific CD8 T cell responses may still be beneficial in both preventative and therapeutic HIV-1 vaccines.

  • Two conserved amino acids within the NSs of SFTS phlebovirus are essential for anti-interferon activity [PublishAheadOfPrint]

  • The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-bbeta;) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-bbeta; responses, the role of the N-terminal region in antagonising host antiviral responses remains to be defined. Herein, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-bbeta; mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signalling protein (MAVS)-mediated activation of IFN-bbeta; promoter activity. Although the SFTSV NSs did not prevent ubiquitination of TBK1, they associate with TBK1 though its N-terminal kinase domain (residues 1-307) to block the autophosphorylation of TBK1. Further, we found that both wild-type and 21/23A mutant NSs of SFTSV suppressed the NLRP3 inflammasome-dependent IL-1bbeta; secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signalling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.

    Importance

    Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection, but also in initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294 amino acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-bbeta;) production and enhance viral replication. Herein, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-bbeta; production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

  • Middle East respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral entry into target cells [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. Here, we re-examined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK not only inhibited the enzymatic activity of furin but also that of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells, and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.

    IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be re-examined carefully. Here, we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

  • Expression of the vaccinia virus anti-apoptotic F1 protein is blocked by PKR in the absence of the viral E3 protein. [PublishAheadOfPrint]

  • Poxviruses encode many proteins with the ability to regulate cellular signaling pathways. One such protein is the vaccinia virus innate immunity modulator E3. Multiple functions have been ascribed to E3, including modulating the cellular response to double stranded RNA, inhibiting the NF-B and IRF3 pathways, and dampening apoptosis. Apoptosis serves as a powerful defense against damaged and unwanted cells and is an effective defense against viral infection; many viruses therefore encode proteins that prevent or delay apoptosis. Here we present data indicating that E3 does not directly inhibit the intrinsic apoptotic pathway; instead, it suppresses apoptosis indirectly by stimulating expression of the viral F1 apoptotic inhibitor. Our data demonstrate that E3 promotes F1 expression by blocking activation of the double stranded RNA activated protein kinase R (PKR). F1 mRNA is present in cells infected with E3-null virus but the protein product does not detectably accumulate, suggesting a block at the translational level. We also show that two 3' co-terminal transcripts span the F1 ORF, a situation previously described for the vaccinia mRNAs encoding the J3 and J4 proteins. One of these is a conventional monocistronic transcript of the F1L gene, while the other arises by read-through transcription from the upstream F2L gene and does not give rise to appreciable levels of F1 protein.

    IMPORTANCE Previous studies have shown that E3-deficient vaccinia virus triggers apoptosis of infected cells. Our study demonstrates that this pro-apoptotic phenotype stems at least in part from the failure of the mutant virus to produce adequate quantities of the viral F1 protein, which acts at the mitochondria to directly block apoptosis. Our data establish a regulatory link between the vaccinia virus proteins that suppress the innate response to double stranded RNA and those that block the intrinsic apoptotic pathway.

  • Herpes simplex virus 1 UL36USP antagonizes type I IFN-mediated antiviral innate immunity [PublishAheadOfPrint]

  • Type I interferons (IFNs), as major components of the innate immune system, play a vital role in host resistance to a variety of pathogens. Canonical signaling mediated by type I IFNs activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway through binding to IFN-aalpha;/bbeta; receptor (IFNAR), resulting in transcription of IFN-stimulated genes (ISGs). However, viruses have evolved multiple strategies to evade this process. Here, we report that herpes simplex virus 1 (HSV-1) ubiquitin-specific protease (UL36USP) abrogates type I IFN-mediated signaling pathway independent of its deubiquitinase (DUB) activity. In this study, ectopically expressed UL36USP inhibited IFN-bbeta; induced activation of ISRE promoter and transcription of ISGs. And overexpression of UL36USP lacking DUB activity did not affect this effect. Furthermore, UL36USP was demonstrated to antagonize IFN-bbeta; induced activation of JAKs and STATs via specifically binding to the IFNAR2 subunit and blocking the interaction between JAK1 and IFNAR2. More importantly, knockdown of HSV-1 UL36USP restored the formation of JAK1-IFNAR2 complex. These findings underline the roles of UL36USP-IFNAR2 interaction in counteracting type I IFN-mediated signaling pathway and reveal a novel evasion mechanism of antiviral innate immunity by HSV-1.

    IMPORTANCE Type I interferons (IFNs) mediate transcription of numerous antiviral genes, creating a remarkable antiviral state in the host. Viruses have evolved various mechanisms to evade this response. Our results indicated that herpes simplex virus 1 (HSV-1) encoded ubiquitin-specific protease (UL36USP) as an antagonist to subvert type I IFN-mediated signaling. UL36USP was identified to significantly inhibited IFN-bbeta;-induced signaling independent of its deubiquitinase (DUB) activity. The underlying mechanism of UL36USP antagonizing type I IFN-mediated signaling was to specifically bind with IFNAR2 and disassociate JAK1 from IFNAR2. For the first time, we identify UL36USP as a crucial suppressor for HSV-1 to evade type I IFN-mediated signaling. It also provides new insights into the innate immune evasion by HSV-1 and will facilitate our understanding about host-virus interplay.

  • Mutation W222L at the receptor binding site of hemagglutinin could facilitate viral adaption from equine influenza A(H3N8) virus to dogs [PublishAheadOfPrint]

  • An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then, has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substituation of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We've demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., aalpha; 2,3-linkage or aalpha;2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.

    Significance statement Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., aalpha;2,3-linked and aalpha;2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

  • Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells [PublishAheadOfPrint]

  • We are pursuing cancer immunotherapy with neuro-attenuated recombinant poliovirus, PVSRIPO. PVSRIPO is the live attenuated type 1 (Sabin) poliovirus vaccine carrying a heterologous internal ribosomal entry site (IRES) of human rhinovirus type 2 (HRV2). Intratumoral infusion of PVSRIPO is showing promise in the therapy of recurrent WHO grade IV malignant glioma (glioblastoma), a notoriously treatment-refractory cancer with dismal prognosis. PVSRIPO exhibits profound cytotoxicity in infected neoplastic cells expressing the poliovirus receptor CD155. In addition, it elicits intriguing persistent translation and replication giving rise to sustained type I interferon (IFN)-dominant proinflammatory stimulation of antigen presenting cells. A key determinant of the inflammatory footprint generated by neoplastic cell infection and its role in shaping the adaptive response after PVSRIPO tumor infection, is the virus' inherent relationship to the host's innate antiviral response. In this report, we define subversion of innate host immunity by PVSRIPO, enabling productive viral translation and cytopathogenicity with extremely low multiplicities of infection in the presence of an active innate antiviral IFN response.

    IMPORTANCE Engaging innate antiviral responses is considered key for instigating tumor-antigen specific antitumor immunity with cancer immunotherapy approaches. However, they are a double-edged sword for attempts to enlist viruses in such approaches. In addition to their role in the transition from innate to adaptive immunity, innate antiviral IFN responses may intercept the viral life cycle in cancerous cells, prevent viral cytopathogenicity, and restrict viral spread. This has been shown to reduce overall anti-tumor efficacy of several proposed oncolytic virus prototypes, presumably by limiting direct cell killing and the ensuing inflammatory profile within the infected tumor. In this report, we outline how an unusual recalcitrance of polioviruses towards innate antiviral responses permits viral cytotoxicity and propagation in neoplastic cells, combined with engaging active innate antiviral IFN responses.

  • Pol-driven replicative capacity impacts disease progression in HIV-1 subtype C infection [PublishAheadOfPrint]

  • CD8+ T cell mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a GFP-reporter T-cell assay. In recently-infected individuals, RT-integrase driven RC correlated significantly with viral load set point (r = 0.25; p = 0.03) and CD4+ T cell decline (p = 0.013). Moreover, significant associations between RT-integrase driven RC and viral load (r = 0.28; pllt;0.0001) and CD4+ T cell count (r =-0.29; pllt;0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (p = 0.05), as was expression of HLA-B*07 (p = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (p=0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally-described HLA-restricted CTL epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant, and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC.

    IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein but the impact of Pol function on HIV disease progression is not well-defined. Here we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C infected individuals with recent and chronic infection and measured their ability to replicate in vitro. We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.

  • In vivo labelling of adenovirus DNA identifies chromatin anchoring and biphasic genome replication. [PublishAheadOfPrint]

  • Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study we used a bacterial partitioning system based on the AnchOR3 technology to establish a versatile in vivo imaging system for adenoviral genomes. This system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determined replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced post-replication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo. This system is able to identify novel spatio-temporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double stranded DNA phase.

    IMPORTANCE Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here we integrated the AnchOR3 system, an in vivo DNA tagging technology, into the adenoviral genome for real time genome detection. AnchOR3-tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cellr-to-cell transfer. We characterize the spatio-temporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The AnchOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.

  • Human cytomegalovirus tropism modulator UL148 interacts with SEL1L, a cellular factor that governs ER-associated degradation of the viral envelope glycoprotein, gO [PublishAheadOfPrint]

  • UL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into mechanism, we used mass spectrometry to identify proteins that co-immunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected with a UL148-null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, siRNA silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO, but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERAD substrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery, and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism.

    IMPORTANCE. Viral glycoproteins in large part determine the cell types that an enveloped virus can infect, and hence play crucial roles in transmission and pathogenesis. The glycoprotein H/L heterodimer (gH/gL) is part of the conserved membrane fusion machinery that all herpesviruses use to enter cells. In human cytomegalovirus (HCMV), gH/gL participates in alternative complexes in virions, one of which is a trimer of gH/gL with glycoprotein O (gO). Here, we show that gO is constitutively degraded during infection by the endoplasmic reticulum-associated degradation (ERAD) pathway, and that UL148, a viral factor that regulates HCMV cell tropism, interacts with the ERAD machinery and slows gO decay. Since gO is required for cell-free virus to enter new host cells, but dispensable for cell-associated spread that resists antibody neutralization, our findings imply that the post-translational instability of a viral glycoprotein provides a basis for viral mechanisms to modulate tropism and spread.

  • Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering [PublishAheadOfPrint]

  • Morbilliviruses (e.g. Measles virus (MeV) or Canine Distemper Virus (CDV)) host cell entry is coordinated by two interacting envelope glycoproteins; namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H-proteins consists of stalk, connector and head domains that assemble into functional non-covalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed "linker") and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a "non-conservative" mutagenesis-scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids, prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to SLAM/CD150 receptor. Non-denaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H-tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H-tetramerization per se was not the only mechanism preventing F-activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion-promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine-substitution of natural cysteines (H-C139A and H-C154A), thus unable to form covalently-linked tetramers, were fusion activation-defective. Our data suggest a dual-regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.

    IMPORTANCE Measles virus (MeV) and canine distemper virus (CDV) remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). Current hypothesis postulates that tetrameric H-ectodomains (composed of stalk, connector and head domains) may undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis-scan analysis of both microdomains revealed that substituting amino acid 146 in the H-linker region with non-hydrophobic residues produced covalent H-tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional "pre-F-triggering" H-tetramers. Furthermore, such structures might be critical to convert receptor engagement into F-activation.

  • Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression [PublishAheadOfPrint]

  • Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors, [vTFs]) supporting late gene expression were conserved in bbeta;- and -herpesviruses, but not in aalpha;-herpesviruses. Here, we performed coimmunopreciptation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics, and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex, and the pM91-pM79 interaction is essential for viral late gene expression.

    IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex; (ii) demonstrated that pM91 interacts with pM79, and that they colocalize in the nuclear viral replication compartments; (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication; and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation how CMV regulates late gene expression, and have important implications for the design of antiviral strategies.

  • Epstein-Barr Virus nuclear antigen 3C facilitates cell proliferation by regulating Cyclin D2 [PublishAheadOfPrint]

  • Cell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. EBV-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes Cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to Cyclin D2, and co-localizes together in nuclear compartments. We show that EBNA3C regulated the promoter of Cyclin D2 through cooperation with master transcription factor Bcl6 and enhanced its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of Cyclin D2, suggesting that Cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors which drives the oncogenic process.

    IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb cell cycle process thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes Cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to Cyclin D2 and co-localizes together in nuclear compartments. EBNA3C enhances Cyclin D2 stability by inhibiting its ubiquitin-dependent degradation, and significantly promotes cell proliferation in the presence of Cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating Cyclin D2 protein and raising the possibility of developing new anti-cancer therapies against EBV-associated cancers.

  • Nup153 Unlocks the Nuclear Pore Complex for HIV-1 Nuclear Translocation in Non-dividing Cells [PublishAheadOfPrint]

  • HIV-1 displays the unique ability to infect non-dividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect non-dividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not non-dividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG-repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and non-dividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not non-dividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups) such as Nup153 with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes, in a manner resembling that of HIV-1 in CPSF6 knockout cells, or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect non-dividing cells. Additionally, the observed viral integration profiles suggested that integration site-selection is a multi-parameter process that depends upon nuclear factors and the state of the cellular chromatin.

    IMPORTANCE One of the key advantages that distinguish lentiviruses such as HIV-1 from all other retroviruses, is its ability to infect non-dividing cells. Interaction of the HIV-1 capsid with Nup153 andCPSF6 is important for nuclear entry and integration; however, it is not clear the contribution of each of these proteins to nuclear import and integration. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in non-dividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site-selection is a multi-parameter process that depends upon nuclear factors and the state of the cellular chromatin.

  • Structural Comparison of Human Anti-HIV-1 gp120 V3 MAbs of the Same Gene Usage Induced by Vaccination and Chronic Infection [PublishAheadOfPrint]

  • Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (mAbs) encoded by the same immunoglobulin genes in different stages of maturation can help to understand the maturation process. We have analyzed four human anti-V3 mAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two mAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime - protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced mAbs are lower than in chronic HIV infection-induced mAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these mAbs bind the V3 epitope with a new cradle-binding mode, and that the V3 bbeta;-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germline sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germline sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.

    IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 mAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germline sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.

  • SIV persistence in cellular and anatomic reservoirs in ART-suppressed infant rhesus macaques [PublishAheadOfPrint]

  • Worldwide, nearly two million children are infected with HIV, with breastfeeding accounting for the majority of contemporary HIV transmissions. Antiretroviral therapy (ART) has reduced HIV-related morbidity and mortality but is not curative. The main barrier to a cure is persistence of latent HIV in long-lived reservoirs. However, our understanding of the cellular and anatomic sources of the HIV reservoir during infancy and childhood is limited. Here, we developed a pediatric model of ART suppression in orally SIV-infected rhesus macaque (RM) infants, with measurement of virus persistence in blood and tissues after 6-9 months of ART. Cross-sectional analyses were conducted to compare SIV RNA and DNA levels in adult and infant RMs naïve to treatment and on ART. We demonstrate efficient viral suppression following ART initiation in SIV-infected RM infants with sustained undetectable plasma viral loads in the setting of heterogeneous penetration of ART into lymphoid and gastrointestinal tissues and low drug levels in the brain. We further show reduction in SIV RNA and DNA on ART in lymphoid tissues of both infant and adult RMs, but stable (albeit low) levels of SIV RNA and DNA in the brains of viremic and ART-suppressed infants. Finally, we report a large contribution of naïve CD4+ T-cells to the total CD4 reservoir of SIV in blood and lymph nodes of ART-suppressed RM infants, that differs from what we show in adults. These results reveal important aspects of HIV/SIV persistence in infants and provide insight into strategic targets for cure interventions in a pediatric population.

    IMPORTANCE While antiretroviral therapy (ART) can reduce HIV replication, the virus cannot be eradicated from an infected individual and our incomplete understanding of HIV persistence in reservoirs greatly complicates the generation of a cure for HIV. Given the immaturity of the infant immune system, it is of critical importance to study HIV reservoirs specifically in this population. Here, we established a pediatric animal model to simulate breastfeeding transmission and study SIV reservoirs in rhesus macaques (RM) infants. Our study demonstrates that ART can be safely administered to infant RM for prolonged periods of time and efficiently controls viral replication in this model. SIV persistence was shown in blood and tissues with a similar anatomic distribution of SIV reservoirs in infant and adult RMs. However, in the peripheral blood and lymph nodes, a higher contribution of the naïve CD4+ T-cells to the SIV reservoir was observed in infants compared to adults.

  • Transduction with lentiviral vectors altered the expression profile of host microRNAs [PublishAheadOfPrint]

  • RNA interference (RNAi) is widely used in gene-knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNA), lentiviral short-hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by miRNA microarray analysis that this inhibition was induced by the alteration of host microRNA (miRNA) expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, 216b-5p, 217, and 30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the up-regulation of JUN contributed to the dysregulation of miR-216a-5p, 216b-5p, and 217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

    IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs over-saturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knock out studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which, in turn, up-regulated miR-216a-5p, miR-216b-5p, and miR-217. MiR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

  • Cell-derived viral genes evolve under stronger purifying selection in Rhadinoviruses [PublishAheadOfPrint]

  • Like many other large dsDNA viruses, herpesviruses are known to capture host genes to evade host defenses. Little is known about the detailed natural history of such genes, nor do we fully understand their evolutionary dynamics. A major obstacle is that they are often highly divergent, maintaining very low sequence similarity to host homologs. Here, we use the herpesvirus genus Rhadinovirus as a model system to develop an analytical approach that combines complementary evolutionary and bioinformatic techniques, offering results that are both detailed and robust for a range of genes. Using a systematic phylogenetic strategy, we identify the original host lineage of viral genes with high confidence. We show that although host immunomodulatory genes evolve rapidly compared to other host genes, they undergo a clear increase in purifying selection once captured by a virus. To characterize this shift in detail, we developed a novel technique to identify changes in selection pressure that can be attributable to particular domains. These findings will inform us on how viruses develop strategies to evade the immune system, and our synthesis of techniques can be reapplied to other viruses or biological systems with similar analytical challenges.

    Importance Viruses and hosts have been shown to capture genes from one another as part of the evolutionary arms-race. Such genes offer a natural experiment of the effects on evolutionary pressure, since the same gene exists in vastly different selective environments. However, viral homologs often bear little similarity to the original sequence, complicating the reconstruction of their shared evolutionary history with host counterparts. In this study, we use a genus of herpesviruses as a model system to thoroughly investigate the evolution of host-derived viral genes, using a synthesis of genomics, phylogenetics, selection analysis as well as nucleotide and amino acid modeling.

  • Characterization of the plasmacytoid dendritic cell response to transmitted/founder and non-transmitted variants of HIV-1 [PublishAheadOfPrint]

  • HIV-1 infection often arises from a single transmitted/founder (TF) viral variant, amongst a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically non-dominant in blood and genital secretions, indicating unique traits. The plasmacytoid dendritic cell (pDC) is the primary IFN-aalpha; producing cell in response to viral infections, and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of the pDC on transmission is unknown. We investigated whether evasion of pDC responses was a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three non-transmitted (NT) variants from five transmission linked donor-pairs and secretion of IFN-aalpha; and TNF-aalpha; was measured by ELISA. No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite trending towards enhanced IFN-aalpha; and TNF-aalpha; in response to TF viruses. NT viruses demonstrated polarization towards production of either IFN-aalpha; or TNF-aalpha;, indicating possible dysregulation. Also, for NT viruses, IFN-aalpha; secretion was associated with increased resistance of the virus to inactivation to IFN-aalpha; in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation, compared to non-transmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1.

    IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell recruited to the site of HIV-1 exposure, however its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor-pair linked transmitted/founder and non-transmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1 viruses, instead of suppressing pDC responses, induced IFN-aalpha; and TNF-aalpha; secretion at comparable levels to viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-aalpha; and TNF-aalpha; production as well as a strong relationship between IFN-aalpha; secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to non-transmitted HIV variants. pDC may, however, be important drivers of viral evolution in vivo.

  • Extracellular vesicles released by herpes simplex virus 1 infected cells block virus replication in recipient cells in a STING-dependent manner. [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) infected cells release extracellular vesicles that deliver to uninfected cells viral factors and host components such as the stimulator of interferon genes (STING) which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1 infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING along with the EV markers CD63 and CD9 were found in light density fractions while HSV components accumulated in heavy density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV-donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1 infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1 infected cells negatively impact the infection and could control the dissemination of the virus.

    IMPORTANCE Extracellular vesicles are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1 infected cells carry innate immune components such as STING and other host and viral factors, they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1 infected cells could control HSV-1 dissemination promoting its persistence in the host.

  • Roles of the Phosphorylation of Herpes Simplex Virus 1 UL51 at a Specific Site in Viral Replication and Pathogenicity [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184, (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mis-localized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivo.

    IMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated, and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread and nuclear egress in cell cultures, and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.

  • Engineered Dengue Virus Domain III Proteins Elicit Cross-neutralizing Antibody Responses in Mice [PublishAheadOfPrint]

  • Dengue virus is the most globally prevalent mosquito-transmitted virus. Primary infection with one of four co-circulating serotypes (DENV1-4) causes a febrile illness but secondary infection with a heterologous serotype can result in severe disease due in part to antibody dependent enhancement of infection (ADE). In ADE, cross-reactive but non-neutralizing antibodies, or sub-protective levels of neutralizing antibodies, promote uptake of antibody-opsonized virus in Fc- receptor-positive cells. Thus, elicitation of broadly neutralizing antibodies (bNAbs), but not non-neutralizing antibodies, is desirable for Dengue vaccine development. Domain III of the envelope glycoprotein (EDIII) is targeted by bNAbs and thus an attractive immunogen. However, immunization with EDIII results in sera with limited neutralization breadth. We developed "resurfaced" EDIII immunogens ("rsDIIIs") whereby the A/G-strand epitope that is targeted by bNAb 4E11 is maintained, but less desirable epitopes are masked. RsDIIIs bound 4E11, but not serotype-specific or non-neutralizing antibodies. One rsDIII and, unexpectedly, WT DENV-2 EDIII elicited cross-neutralizing antibody responses against DENV1-3 in mice. While these sera were cross-neutralizing, they were not sufficiently potent to protect AG129 immunocompromised mice at a dose of 200 mmu;L (FRNT50 at titer of ~1:60-1:130) against mouse adapted DENV-2. Our results provide insight into immunogen design strategies based on EDIII.

    IMPORTANCE Dengue virus causes approximately 390 million infections per year. Primary infection by one serotype causes a self-limiting febrile illness, but secondary infection by a heterologous serotype can result in Severe Dengue, which is characterized by hemorrhagic fever and shock syndrome. This severe disease is thought to arise because of cross-reactive, non- or poorly-neutralizing antibodies from the primary infection, that are present in serum at the time of secondary infection. These cross-reactive antibodies enhance the infection rather than controlling it. Therefore, induction of a broadly and potently neutralizing antibody response is desirable for Dengue vaccine development. Here we explore a novel strategy developing immunogens based on domain III of the E glycoprotein, where undesirable epitopes (non-neutralizing, or non-conserved) are masked by mutation. This work provides fundamental insight into the immune response to domain III that can be leveraged for future immunogen design.

  • DNA polymerase sequences of New World monkey cytomegaloviruses: another molecular marker to infer Platyrrhini systematics [PublishAheadOfPrint]

  • Over the past few decades, a large number of studies have identified herpesvirus sequences from many mammalian species around the world. Among the different non-human primate species tested so far for cytomegaloviruses, only a few were from the New World. Seeking to identify cytomegalovirus homologues in New World monkeys (NWMs), we carried out molecular screening of 244 blood DNA samples from 20 NWM species from Central and South America. Our aim was to reach a better understanding of their evolutionary processes within the Platyrrhini parvorder. Using polymerase chain reaction amplification with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase gene, we characterized novel viral sequences from 12 species belonging to seven genera representative of the three NWM families. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Cytomegalovirus genus. Previously determined host taxa allowed us to demonstrate a good correlation between the distinct monophyletic clades of viruses with those of the infected primates at the genus level. In addition, the evolutionary branching points that separate NWM CMVs were congruent with the divergence dates of their hosts at the genus level. These results significantly expand our knowledge of the host range of this viral genus and strongly support the co-speciation between these viruses and their hosts. In this respect, we propose that NWM CMV DNA polymerase sequences could serve as a reliable molecular marker to infer Platyrrhini phylogenetics.

    IMPORTANCE Investigating evolutionary processes between viruses and non-human primates has led to the discovery of a high number of herpesviruses. No study published so far on primate cytomegaloviruses has extensively studied New World monkeys (NWMs) at the subspecies, species, genus and family levels. The present study sought to identify cytomegalovirus homologues in NWMs and decipher their evolutionary relationships. This led us to characterize novel viruses in 12 of the 20 primate species tested representative of the three NWM families. The identification of distinct viruses in these primates not only significantly expands our knowledge of the host range of this viral genus, but has also shed light on their evolutionary history. Phylogenetic analyses and molecular dating of the sequences obtained support a virusnndash;host coevolution.

  • CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals [PublishAheadOfPrint]

  • A recent study conducted in blood has proposed CD32 as the marker identifying the llsquo;elusive' HIV reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes (LNs) of HIV-1 uninfected subjects, viremic untreated and long-term treated HIV-1 infected individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells was increased in viremic as compared to treated individuals in LNs and a large proportion (up to 50%) of CD32+ cells co-expressed PD-1 and were enriched within T follicular helper cells (Tfh) cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+ and PD-1+ CD4 T cells as compared to CD32- and PD-1- cells in both viremic and treated individuals but there was no difference between CD32+ and PD-1+ cells. There was not enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in ART treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+PD-1+ CD4 T cells were significantly enriched in cell associated HIV RNA as compared to CD32-PD-1- (average 5.2 fold in treated and 86.6 fold in viremics), to CD32+PD-1- (2.2 fold in treated and 4.3 fold in viremics) and to CD32-PD-1+ cell populations (2.2 fold in ART treated and 4.6 fold in viremics). Similar levels of HIV-1 transcription were found in CD32+PD-1- and CD32-PD-1+ CD4 T cells. Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and CD32 and PD-1 co-expression the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.

    IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells co-expressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

  • Ancient evolutionary origin and positive selection of the retroviral restriction factor Fv1 in muroid rodents [PublishAheadOfPrint]

  • The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirusnndash;L (MuERV-L) family of ERVs suggests that Fv1 was co-opted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the Mus genus. Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside of the genus Mus. We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1. Our analysis also reveals that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the Mus genus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae. Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs.

    IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, the Fv1 gene found in mice, was thought to have originated in the genus Mus. In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum insertion time of 45 million years into the genome of rodents for the ancestral progenitor of Fv1. While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular "arms race" in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been co-evolving with unidentified retroviruses for millions of years.

  • The apical region of the herpes simplex virus major capsid protein promotes capsid maturation [PublishAheadOfPrint]

  • The herpesvirus capsid assembles in the nucleus as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown, but it is dependent upon activation of the VP24 internal protease. Scaffold cleavage triggers angularization of the shell and its decoration with the VP26 and pUL25 capsid-surface proteins. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein (VP5) is surface exposed. We investigated whether the VP5 apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively-charged amino acids in the apical region contribute to VP26 acquisition. To our surprise neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that these clusters consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation.

    IMPORTANCE Herpesviruses assemble capsids and encapsidate their genomes by a process that is unlike other mammalian viruses but is similar to some bacteriophage. Many important aspects of herpesvirus morphogenesis remain enigmatic, including how the capsid shell matures into a stable angularized configuration. Capsid maturation is triggered by activation of a protease that cleaves an internal protein scaffold. We report on the fortuitous discovery that a region of the major capsid protein that is exposed on the outer surface of the capsid also contributes to capsid maturation, demonstrating that the morphogenesis of the capsid shell from its procapsid precursor to the mature angularized form is dependent upon internal and external components of the megastructure.

  • Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters [PublishAheadOfPrint]

  • How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing co-localization at an integrated lacO array of fusions of lacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP and Pol II across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and 27 acetylation at the E2early, E3, and E4 promoters, and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters, but was required for E4 transcription at a step subsequent to Pol II pre-initiation complex assembly.

    Importance: Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone post-translational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300 as reported by Pelka et al. 2009, and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

  • The 5' untranslated region of the capsid protein 2 gene of mink enteritis virus is essential for its expression [PublishAheadOfPrint]

  • Mink enteritis virus (MEV), as a parvovirus, is among the smallest of the animal DNA viruses. The limited genome leads to multifunctional sequences and complex gene expression regulation. Here we show that the expression of viral capsid protein 2 (VP2) of MEV requires its 5' untranslated regions (5' UTR) which promote VP2 gene expression at both transcriptional and translational levels. The expression of VP2 was inhibited in several common eukaryotic expression vectors. Our data showed that the 5' UTR of VP2 enhanced capsid gene transcription but not increased stability or promotes nucleo-cytoplasmic export of VP2 mRNA. Analysis of the functions of 5' UTR fragments showed that the proximal region (1-270 nt, +1 to +270 relative to the transcription initiation site, 2048-2317 nt of MEV-L) of 5' UTR of VP2 was necessary for VP2 transcription, and also promoted the activity of P38 promoter. Unexpectedly, further analysis showed that deletion of the distal region (271-653 nt) of the 5' UTR of VP2 almost completely abolished VP2 translation in the presence of P38 whereas the transcription was still induced significantly. Furthermore, using a luciferase reporter bicistronic system, we identified that the 5' UTR had an internal ribosome entry site (IRES)-like function which could be enhanced by NS1 via 382-447 nt site. Mutation of the 5' UTR in the MEV full-length clones further showed that the 5' UTR was required for VP2 gene expression. Together, our data reveal an undiscovered function of 5' UTR of MEV VP2 in regulating viral gene expression.

    Importance MEV, a parvovirus, causes acute enteritis in mink. In the present report, we describe an untranslated sequences-dependent mechanism by which MEV regulates capsid gene expression. Our results highlight the roles of untranslated sequences in regulating the transcriptional activity of P38 promoter and translation of capsid genes. These data also reveal the possibility of an unusual translation mechanism in capsid proteins expression and the multiple functions of non-structural protein. A better understanding of the gene expression regulation mechanism of this virus will help to design new vaccines and targets for antiviral agents against MEV.

  • PARP1 Stabilizes CTCF Binding and Chromatin Structure to Maintain Epstein Barr Virus Latency Type [PublishAheadOfPrint]

  • Epstein Barr Virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is post-translationally modified by the host enzyme PARP1. PARP1, or Poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome, and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency, and could ultimately contribute to an EBV-specific treatment strategy for AIDS-related or post-transplant lymphomas.

    IMPORTANCE Epstein Barr Virus (EBV) is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome, and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically non-pathological; however, latent infection is potentially oncogenic, and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and post-transplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

  • Crystal structure of the dimerized N-terminus of porcine circovirus type 2 replicase protein reveals a novel antiviral interface [PublishAheadOfPrint]

  • Two replicase (Rep) proteins, Rep and Rep', are encoded by porcine circovirus (PCV) ORF1; Rep is a full ORF1 transcript and Rep' is a truncated transcript generated by splicing. These two proteins are crucial for the rolling-circle replication (RCR) of PCV. The N-terminal sequences of Rep and Rep' are identical and interact to form homo- or hetero-dimers. The three types of dimers perform different functions during replication. A structural examination of the interfacing termini has not been performed. In this study, a crystal structure of dimerized Rep protein N-termini was resolved at 2.7 AAring;. The dimerized protein was maintained by nine intermolecular hydrogen bonds and fifteen pairs of hydrophobic interactions. The amino acid residue Ile37 participates in eleven of the hydrophobic interactions mostly with its side chain. To find the predominant sites for protein dimerization and virus replicantion, a series of mutant proteins and virus replicons were generated by alanine substitution. Of all the single amino acid substitutions, the mutation at Ile37 showed the greatest effect on the protein dimerization and virus replication. A double mutation at Leu35 and Ile37 almost eliminated the protein dimerization, and had the greatest negative effect on virus replication. These studies demonstrate that Leu35 and Ile37 are the most important residues for protein dimerization and crucial for virus replication. Our results also show that PCV replication can be decreased by disrupting the dimerization of Rep or Rep' at their N-termini, suggesting that the structural interface responsible for dimerization offers a promising antiviral target.

    IMPORTANCE Porcine circovirus type 2 (PCV2) is one of the most economically damaging pathogens affecting the swine industry. Although vaccines have been available for more than ten years, the virus still remains prevalent. More effective strategies for disease prevention are clearly required. The Rep and Rep' proteins of the virus have identical N-terminal regions that interact with each other allowing the formation of homo- or hetero-dimers. The hetero-dimer has crucial functions during different stages of viral replication. Here, we resolved the crystal structure of the Rep (Rep') dimerization domain. The individual residues involved in the intermolecular interaction were visualized in the protein structure, and several interactions were verified by mutant analysis. Our studies show that disrupting the interaction decreases viral replication, thus revealing a new target for the design of antiviral agents.

  • Feline leukemia virus disease outcomes in a domestic cat breeding colony: Relationship to endogenous FeLV and other chronic viral infections [PublishAheadOfPrint]

  • Exogenous feline leukemia virus (FeLV) is a feline Gammaretrovirus that results in a variety of disease outcomes. Endogenous FeLV (enFeLV) is a replication-defective provirus found in species belonging to the Felis genus, which includes the domestic cat (Felis catus). There have been few studies examining interaction between enFeLV genotype and FeLV progression. We examined point-in-time enFeLV and FeLV viral loads, as well as occurrence of FeLV/enFeLV recombinants (FeLV-B), to determine factors relating to clinical disease in a closed breeding colony of cats during a natural infection of FeLV. Coinfections with feline foamy virus (FFV), feline gammaherpesvirus (FcaGHV-1), and feline coronavirus (FCoV) were also documented and analyzed for impact on cat health and FeLV disease. Correlation analysis and structural equation modeling techniques were used to measure interactions among disease parameters. Progressive FeLV disease and FeLV-B presence were associated with higher FeLV proviral and plasma viral loads. Female cats were more likely to have progressive disease and FeLV-B. Conversely, enFeLV copy number was higher in male cats and negatively associated with progressive FeLV disease. Males were more likely to have abortive FeLV disease. FFV proviral load was found to correlate positively with higher FeLV proviral and plasma viral load, detection of FeLV-B, and FCoV status. Male cats were much more likely to be infected with FcaGHV-1 than female cats. This analysis provides insights into the interplay between endogenous and exogenous FeLV during naturally occurring disease, and reveals striking variation in the infection patterns among four chronic viral infections of domestic cats.

    Importance Endogenous retroviruses are harbored by many animals and their interaction with exogenous retroviral infections have not been widely studied. Feline leukemia virus (FeLV) is a relevant model system to examine this question, as endogenous and exogenous forms of the virus exist. In this analysis of a large domestic cat breeding colony naturally infected with FeLV, we document that enFeLV copy number was higher in males and inversely related to FeLV viral load and associated with better FeLV disease outcomes. Females had lower enFeLV copy number and were more likely to have progressive FeLV disease and FeLV-B subtypes. FFV viral load was correlated with FeLV progression. FFV, FcaGHV-1, and FeLV displayed markedly different patterns of infection with respect to host demographics. This investigation reveals complex coinfection outcomes and viral ecology of chronic infections in a closed population.

  • Localized induction of wild-type and mutant alpha-synuclein aggregation reveals propagation along neuroanatomical tracts [PublishAheadOfPrint]

  • Misfolded aalpha;-synuclein (aalpha;S) may exhibit a number of characteristics similar to the prion protein, including the apparent ability to spread along neuroanatomical connections. The demonstration for this mechanism of spread is largely based on the intracerebral injections of pre-aggregated aalpha;S seeds in mice, in which it cannot be excluded that diffuse, surgical perturbations and hematogenous spread also contribute to the propogation of pathology. For this reason, we have utilized the sciatic nerve as a route of injection to force the inoculum into the lumbar spinal cord and induce a localized site for the onset of aalpha;S inclusion pathology. Our results demonstrate that mouse aalpha;S fibrils injected unilaterally in the sciatic nerve are efficient in inducing pathology and the onset of paralytic symptoms in both the M83 and M20 lines of aalpha;S transgenic mice. In addition, a spatiotemporal study of these injections revealed a predictable spread of pathology to brain regions whose axons synapse directly on ventral motor neurons in the spinal cord, strongly supporting axonal transport as a mechanism of spread of the aalpha;S inducing, or seeding, factor. We also revealed a relatively decreased efficiency for human aalpha;S fibs containing the E46K mutation to induce disease via this injection paradigm, supportive of recent studies demonstrating a diminished ability for this mutant aalpha;S to undergo aggregate induction. These results further demonstrate prion-like properties for aalpha;S by the ability for a progression and spread of aalpha;S inclusion pathology along neuroanatomical connections.

    IMPORTANCE The accumulation of aalpha;-synuclein (aalpha;S) inclusions is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Recently, a number of studies have demonstrated similarities between the prion protein and aalpha;S, including its ability to spread along neuroanatomical tracts throughout the central nervous system (CNS). However, there are caveats in each of these studies in which the injection routes used, had the potential to result in a widespread dissemination of the aalpha;S-containing inocula, making it difficult to precisely define the mechanisms of spread. In this study, we assess the spread of pathology following a localized induction of aalpha;S inclusions in the lumbar spinal cord following a unilateral injection in the sciatic nerve. Using this paradigm, we demonstrate the ability for aalpha;S inclusion spread and/or induction along neuroanatomical tracts within the CNS of two aalpha;S overexpressing mouse models.

  • Upregulation of miR-711 mediates HIV-1 Vpr promotion of KSHV latency and induction of pro-proliferative/survival cytokines by targeting the Notch/NF-{kappa}B signaling axis [PublishAheadOfPrint]

  • Co-infection with HIV-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) often leads to AIDS-related malignancies including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The interaction between HIV and KSHV plays a pivotal role in the progression of these malignancies. We have previously demonstrated that, by upregulating miR-942-5p, HIV-1 viral protein R (Vpr) inhibits KSHV lytic replication by targeting IBaalpha; to activate the NF-B signaling (J Virol, 2016; 90 (19):8739-53). Here we show that Vpr inactivates Notch signaling, resulting in inhibition of KSHV lytic replication and induction of pro-proliferative and -survival cytokines, including IL-2, TIMP-1, IGF-1 and NT-4. Mechanistically, Vpr upregulates miR-711, which directly targets Notch1 3'UTR. Suppression of miR-711 relieved Notch1 and reduced Vpr inhibition of KSHV lytic replication and Vpr induction of pro-proliferation and nndash;survival cytokines, while overexpression of miR-711 exhibited an opposite effect. Finally, overexpression of Notch1 reduced Vpr induction of NF-B activity by promoting IBaalpha; promoter activity. Our novel findings reveal that by up-regulating miR-711 to target Notch1, Vpr silences Notch signaling to activate the NF-B pathway by reducing IBaalpha; expression, leading to inhibition of KSHV lytic replication and induction of pro-proliferation and -survival cytokines. Therefore, the miR-711/Notch/NF-B axis is important in the pathogenesis of AIDS-related malignancies, and could be an attractive therapeutic target.

    IMPORTANCE HIV-1 infection significantly increases the risk of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) in KSHV-infected individuals. Our previous study has shown that HIV-1 Vpr regulates KSHV life cycle by targeting IBaalpha; to activate NF-B signaling through upregulating cellular miR-942-5p. In this study, we have further found that Vpr inactivates Notch signaling to promote KSHV latency and production of pro-proliferation and -survival cytokines. Another Vpr-upregulated cellular microRNA, miR-711, participates in this process by directly targeting Notch1. As a result, Notch1 upregulation of the IBaalpha; promoter activity is attenuated, resulting in reduced levels of IBaalpha; transcript and protein. Overall, these results illustrate an alternative mechanism of HIV-1 Vpr regulation of KSHV latency and aberrant cytokines through the miR-711/Notch/NF-B axis. Our novel findings further demonstrate the role of a HIV-1-secreted regulatory protein on KSHV life cycle and KSHV-related malignancies.

  • Suppressor of Cytokine Signaling (SOCS)1 and SOCS3 Are Stimulated Within the Eye During Experimental Murine Cytomegalovirus Retinitis in Mice With Retrovirus-Induced Immunosuppression [PublishAheadOfPrint]

  • AIDS-related human cytomegalovirus retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. To study mechanisms of this disease, we use a clinically relevant animal model of murine cytomegalovirus (MCMV) retinitis with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics the progression of AIDS in humans. We found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which hinder immune-related signaling by cytokines, including antiviral type I and type II interferons. The present study demonstrates that in the absence of retinal disease, systemic MCMV infection of mice without MAIDS, but not in mice with MAIDS, leads to mild stimulation of splenic SOCS1 mRNA. In sharp contrast, when MCMV is directly inoculated into the eyes of retinitis-susceptible MAIDS mice, high levels of intraocular SOCS1 and SOCS3 mRNA and protein are produced which are associated with significant intraocular upregulation of IFN- and IL-6 mRNA expression. We also show that infiltrating macrophages, granulocytes, and resident retinal cells are sources of intraocular SOCS1 and SOCS3 protein production during development of MAIDS-related MCMV retinitis, and SOCS1 and SOCS3 mRNA transcripts are detected in retinal areas histologically characteristic of MCMV retinitis. Furthermore, SOCS1 and SOCS3 are found in both MCMV-infected cells and uninfected cells, suggesting that these SOCS proteins are stimulated via a bystander mechanism during MCMV retinitis. Taken together, our findings suggest a role for MCMV-related stimulation of SOCS1 and SOCS3 in the progression of retinal disease during ocular, but not systemic, MCMV infection.

    IMPORTANCE Cytomegalovirus infection frequently causes blindness in untreated HIV/AIDS patients. This virus manipulates host cells to dysregulate immune functions and drive disease. Herein we use an animal model of this disease to demonstrate that cytomegalovirus infection within eyes during retinitis causes massive upregulation of immunosuppressive host proteins called suppressors of cytokine signaling (SOCS). As viral overexpression of SOCS proteins exacerbates infection with other viruses, they may also enhance cytomegalovirus infection. Alternatively, the immunosuppressive effect of SOCS proteins may be protective against immunopathology during cytomegalovirus retinitis, and in such a case SOCS mimetics or overexpression treatment strategies might be used to combat this disease. The results of this work therefore provide crucial basic knowledge that contributes to our understanding of the mechanisms of AIDS-related cytomegalovirus retinitis and, together with future studies, may contribute to the development of novel therapeutic targets that could improve the treatment or management of this sight-threatening disease.

  • Probing Zika virus neutralization determinants with glycoprotein mutants bearing linear epitope insertions [PublishAheadOfPrint]

  • Zika virus (ZIKV) glycoproteins are the primary target of the humoral immune response. In this study, we explored the capacity of these glycoproteins to tolerate insertion of linear epitope sequences, and the potential of antibodies that bind these epitopes to inhibit infection. We first created a panel of ZIKV mutants with the FLAG epitope inserted in premembrane (prM) and envelope (E) glycoprotein regions. Insertion locations were based on the results of our recent transposon insertional mutagenesis screen. Although FLAG insertions in prM greatly impaired viral fitness, this sequence was tolerated in numerous surface-exposed E protein sites. We observed that mutants bearing FLAG epitopes in E domains I, II, and the DI-II hinge region were all neutralized by FLAG antibody, however, the neutralization sensitivity varied highly. We measured antibody binding efficiency and found that this closely matched the pattern of neutralization sensitivity. We determined that E glycosylation did not affect antibody binding to a nearby epitope or its capacity of to serve as a neutralization target. Although we could not generate infectious viruses with FLAG epitope insertions in a buried region of E protein domain III, we found that the V5 epitope could be inserted without greatly impacting fitness. Furthermore, this virus was efficiently neutralized by V5 antibodies, highlighting that even buried epitopes can function as neutralization targets. Finally, we analyzed the timing of antibody neutralization activity during cell entry and found that all antibodies blocked a step after cell attachment.

    IMPORTANCE Zika virus (ZIKV) infections are associated with severe birth defects and neurological disease. The structure of the mature ZIKV particle reveals a virion surface covered by the envelope glycoprotein, which is the dominant target of the humoral immune response. It is unclear if all regions of the envelope protein surface, or even llsquo;buriedrrsquo; epitopes, can function as neutralization targets. To test this, we created a panel of ZIKV mutants with epitope insertions in different regions of the envelope protein. In characterizing these viruses, we found that the strength of antibody binding to an epitope is the major determinant of the neutralization potential of an antibody, even a buried region of the envelope protein can be efficiently targeted, and the sole potential envelope glycan does not impact nearby epitope antibody binding and neutralization. Furthermore, this work provides important insights into our understanding of how antibodies neutralize ZIKV.

  • Targeting the HIV-1 spike and co-receptor with bi- and trispecific antibodies for single-component broad inhibition of entry [PublishAheadOfPrint]

  • Protection against acquiring HIV infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMab targeting two epitopes on the major HIV co-receptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98-100% of a 109-virus panel, as well as additivity and potency compared to the individual parental control IgGs. The bispecific molecules designed as tandem single chain variable fragments (scFvs; 10E8fv-N6fv and m36.4-PRO 140fv) displayed a median IC50 of 0.0685 and 0.0131 mmu;g/ml, respectively. A trispecific containing 10E8-PGT121-PGDM1400 Env-specific binding sites was equally potent (median IC50 of 0.0135 mmu;g/ml), while the trispecific molecule targeting Env and CCR5 simultaneously (10E8Fab-PGDM1400fv-PRO 140fv), demonstrated even greater potency with a median IC50 of 0.007 mmu;g/ml. By design, some of these molecules lacked Fc-mediated effector function; therefore, we also constructed a trispecific prototype possessing reconstituted CH2-CH3 domains to restore Fc receptor binding capacity. The molecules developed here, along with those described previously, possess promise as prophylactic and therapeutic agents against HIV.

    IMPORTANCE Broadly neutralizing antibodies (bNAbs) prevent HIV infection in monkey challenge models and suppress HIV replication in infected humans. Combinations of bNAbs are more effective at suppression and antibody-like molecules engineered to have 2 or 3 bNAb combining sites also inhibit HIV replication in monkeys and other animal models. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAb binding sites that recognize the HIV envelope glycoprotein (Env), the HIV co-receptor (CCR5), or that cross-target both Env and CCR5. Several of the bi- and trispecific molecules neutralized most viruses in a diverse cross-clade panel with greater breadth and potency than the individual parental bNAbs. The molecules described here provide additional options to prevent or suppress HIV infection.

  • Vaccination with the conserved caveolin-1 binding motif in HIV-1 glycoprotein gp41 delays the onset of viral infection and provides partial protection in SHIV-challenged cynomolgus macaques [PublishAheadOfPrint]

  • We have previously reported that the CBD1 peptide (SLEQIWNNMTWMQWDK) corresponding to the consensus caveolin-1 binding domain in HIV-1 envelope glycoprotein gp41, elicits peptide-specific antibodies. Herein, we have investigated the cellular immune response, and the protective efficacy against the SHIV162P3 challenge. In addition to the CBD1 peptide, peptides overlapping the caveolin-binding-motif (CBM) (622IWNNMTWMQW631 or 622IWNNMTW628) were fused to a Gag-p24 T helper epitope for vaccination. All immunized cynomolgus macaques responded to a cocktail peptide immunization, by inducing specific T cells and the production of high tittered CBD1/CBM peptide-specific antibodies. Six months after the fourth vaccine boost, six control and five vaccinated animals were challenged weekly by repeated exposure to SHIV162P3 via the mucosal rectal route. All control animals were infected after 1-3 challenges with SHIV, while among the five vaccinated monkeys, three became infected after a delay when compared to control; one was infected after the eighth viral challenge, and one remained uninfected even after the ninth SHIV challenge. Immunized animals maintained a CD4 T cell count and central memory CD4 T cells were less depleted compared to the control group. Furthermore, SHIV challenge stimulates antigen-specific memory T-cell response in vaccinated macaques. Our results indicate that peptides derived from the CBM region can be immunogenic and provide protection against SHIV infection in cynomolgus monkeys.

    IMPORTANCE In HIV-1 producing cells, gp41 exists in a complexed form with caveolin-1, an interaction most probably mediated by the caveolin-1 binding motif. This sequence is highly conserved in every single HIV-1 isolate, thus suggesting that there is constant selective pressure to preserve this sequence for a specific function in the HIV infectious cycle. Consequently, the CBM sequence may represent the "Achilles' heel" of HIV-1 in the development of an efficient vaccine. Our results demonstrate that macaques immunized with the CBM-based peptides displayed a delay in the onset of viral infection and CD4 depletion, as well as a significant induction of antigen-specific memory T-cell response, which is essential for the control of HIV/SIV infections. Finally, as HIV-infected individuals lack anti-CBM immune responses, CBM-based vaccines could have applications as a therapeutic vaccine in AIDS patients.

  • Hepacivirus A Infection in Horses Defines Distinct Envelope Hypervariable Regions and Elucidates Potential Roles of Viral Strain and Adaptive Immune Status in Determining Envelope Diversity and Infection Outcome [PublishAheadOfPrint]

  • Hepacivirus A (also known as non-primate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infection in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A and its roles in immune evasion and persistence are unknown. To address these knowledge gaps we evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals) and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than observed in horses that spontaneously cleared infection (P = 0.0002) or in SCID foals (P llt; 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P llt; 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcome and could be dependent on strain specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank and defined three novel hypervariable regions in E2 at residues 391-402 (HVR1), 450-461 (HVR2) and 550-562 (HVR3). In the experimentally infected horses entropy analysis focused on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that they may be targets of neutralizing antibody and play a role in immune evasion.

    IMPORTANCE HCV is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap we examined envelope gene diversity in horses with resolving and persistent infection. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation was influenced by adaptive immune pressure and may contribute to hepaciviral persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host.

  • Mouse Norovirus infection reduces the surface expression of MHC class I proteins and inhibits CD8+ T cell recognition and activation [PublishAheadOfPrint]

  • Human Noroviruses are highly infectious, single stranded RNA (ssRNA) viruses and the major cause of non-bacterial gastroenteritis worldwide. With the discovery of Murine Norovirus (MNV) and the introduction of an effective model for norovirus infection and replication, knowledge about infection mechanisms and its impact on the host immune response has progressed. A major player in the immune response against viral infections is the group of major histocompatibility complex (MHC) class I proteins, which present viral antigen to immune cells. We have observed that MNV interferes with the antigen presentation pathway in infected cells by reducing the surface expression of MHC class I proteins. We have shown that MNV infected dendritic cells or macrophages have a reduced surface expression of MHC class I proteins compared to uninfected and bystander cells. Transcriptional analysis revealed that this defect is not due to decreased amount of mRNA, but is reflected at the protein level. A defect we have identified to be mediated via the MNV NS3 protein. Significantly, treatment of MNV-infected cells with the endocytic recycling inhibitor Dynasore completely restored the surface expression of MHC class I, whereas treatment with the proteasome inhibitor MG132 partly restored the surface expression of MHC class I. These observations indicate a role of endocytic recycling and proteasome-mediated degradation of the protein. Importantly, we show that due to the reduced surface expression of MHC class I proteins, antigen presentation is inhibited, resulting in the inability of CD8+ T cells to become activated in the presence of MNV infected cells.

    IMPORTANCE Human Noroviruses (HuNoV) are the major cause of non-bacterial gastroenteritis worldwide and cause a great burden on patients and health systems every year. So far, no antiviral treatment or vaccine is available. We show that MNV evades the host immune response by reducing the amount of MHC class I proteins displayed on the cell surface. This reduction leads to a decrease in viral antigen presentation and interferes with the CD8+ T cell response. CD8+ T cells respond to foreign antigen by activating cytotoxic pathways and inducing immune memory to the infection. By evading this immune response MNV is able to replicate efficiently in the host and cells are impaired in their ability to respond to consecutive infections. These findings have a major impact on our understanding of how Noroviruses interact with the host immune response and manipulate immune memory.

  • Preadaptation of SIVsmm facilitated Env-mediated counteraction of human tetherin by HIV-2 [PublishAheadOfPrint]

  • The host restriction factor tetherin inhibits virion release from infected cells and poses a significant barrier to successful zoonotic transmission of primate lentiviruses to humans. While most simian immunodeficiency viruses (SIV), including the direct precursors of HIV-1 and HIV-2, use their Nef protein to counteract tetherin in their natural hosts, they fail to antagonize the human tetherin ortholog. Pandemic HIV-1 group M and epidemic group O strains mastered this hurdle by adapting their Vpu and Nef proteins, respectively, whereas HIV-2 group A uses its envelope (Env) glycoprotein to counteract human tetherin. Whether or how the remaining eight groups of HIV-2 antagonize this antiviral factor has remained unclear. Here, we show that Nef proteins from diverse groups of HIV-2 do not or only modestly antagonize human tetherin, while their ability to down-modulate CD3 and CD4 is highly conserved. Experiments in transfected cell lines and infected primary cells revealed that not only Env proteins of epidemic HIV-2 group A, but also those of a circulating recombinant form (CRF01_AB) and rare groups F and I decrease surface expression of human tetherin and significantly enhance progeny virus release. Intriguingly, we found that many SIVsmm Envs also counteract human as well as smm tetherin. Thus, Env-mediated tetherin antagonism in different groups of HIV-2 presumably stems from a preadaptation of their SIVsmm precursors to humans. In summary, we identified a phenotypic trait of SIVsmm that may have facilitated its successful zoonotic transmission to humans and the emergence of HIV-2.

    Importance HIV-2 groups A to I resulted from nine independent cross-species transmission events of simian immunodeficiency viruses to humans and differ considerably in their prevalence and geographic spread. Thus, detailed characterization of these viruses offers a valuable means to elucidate immune evasion mechanisms and human-specific adaptations determining viral spread. In a systematic comparison of rare and epidemic HIV-2 groups and their simian SIVsmm counterparts, we found that the ability of Nef to down-modulate the primary viral entry receptor CD4 and the T cell receptor CD3 is conserved, while effects on CD28, CD74 and MHC-I surface expression vary considerably. Furthermore, we show that not only the Env proteins of HIV-2 groups A, AB, F and I, but also those of some SIVsmm isolates antagonize human tetherin. This finding helps to explain why SIVsmm has been able to cross the species barrier to humans on at least nine independent occasions.

  • Bub1 Facilitates Virus Entry through Endocytosis in a Model of Drosophila Pathogenesis [PublishAheadOfPrint]

  • In order to establish productive infection and dissemination, viruses usually evolve a number of strategies to hijack and/or subvert the host defense systems. However, host factors utilized by the virus to facilitate infection remain poorly characterized. In this work, we found that Drosophila melanogaster deficient in budding uninhibited by benzimidazoles 1 (bub1), a highly conserved subunit of kinetochores complex regulating chromosome congression (1), became resistant to Drosophila C virus (DCV) infection evidenced in increased survival rates and reduced viral loads, compared to the wild type control. Mechanistic analysis further showed that Bub1 also functioned in the cytoplasm and was essentially involved in clathrin-dependent endocytosis of DCV and other pathogens, thus limiting pathogen entry. DCV infection potentially had strengthened the interaction between Bub1 and the clathrin adaptor on the cell membrane. Furthermore, the conserved function of Bub1 was as well verified in a mammalian cell line. Thus, our data demonstrated a previously unknown function of Bub1 that could be hijacked by pathogens to facilitate their infection and spread.

    IMPORTANCE In this work, we for the first time identify that nuclear protein Bub1 (budding uninhibited by benzimidazoles 1), a highly conserved subunit of kinetochores complex regulating chromosome congression, has a novel and important function on the cell membrane to facilitate the virus to entry host cells. Bub1 deficiency empowers the host to have the ability to resistant viral infection in Drosophila and a human cell line. Bub1 is involved in the virus entry-step through regulating endocytosis. DCV capsid protein can recruit Bub1 and DCV infection can strength the interaction between Bub1 and clathrin-dependent endocytosis component. The restricted entry of VSV and Listeria monocytogenes in bub1 deficient flies and cell lines is also observed. Therefore, our data implicate a previously unknown function of Bub1 that can be hijacked by pathogens to facilitate their entry and Bub1 may serve as a potential antiviral therapy target for limiting viral entry.

  • 25-hydroxycholesterol Production by Interferon Stimulated Gene Cholesterol-25-hydroxylase Restricts Mammalian Reovirus Infection. [PublishAheadOfPrint]

  • Following the initial detection of viral infection, innate immune responses trigger the induction of numerous interferon-stimulated genes (ISGs) to inhibit virus replication and dissemination. One such ISG encodes cholesterol-25-hydroxylase (CH25H), an enzyme that catalyzes the oxidation of cholesterol to form a soluble product, 25-hydroxycholesterol (25HC). Recent studies have found that CH25H is broadly antiviral; it inhibits infection by several viruses. For enveloped viruses, 25HC inhibits membrane fusion, likely by altering membrane characteristics such as hydrophobicity or cholesterol aggregation. However, mechanisms by which 25HC restricts infection of non-enveloped viruses are unknown. We examined whether 25HC restricts infection by mammalian reovirus. Treatment with 25HC restricted infection by reovirus prototype strains type 1 Lang and type 3 Dearing. In contrast to reovirus virions, 25HC did not restrict infection by reovirus infectious subvirion particles, ISVPs, which can penetrate either directly at the cell surface or in early endosomal membranes. Treatment with 25HC altered trafficking of reovirus particles to late endosomes and delayed the kinetics of reovirus uncoating. These results suggest that 25HC inhibits the efficiency of cellular entry of reovirus virions, which may require specific endosomal membrane dynamics for efficient membrane penetration.

    IMPORTANCE

    The innate immune system is crucial for effective responses to viral infection. Type I interferons, central components of innate immunity, induce expression of hundreds of ISGs; however, the mechanisms of action of these antiviral proteins is not well understood. CH25H, an ISG, represents a significant constituent of these cellular antiviral strategies, as its metabolic product, 25HC, can act in both autocrine and paracrine fashion to protect cells from infection, and has been shown to limit viral infection in animal models. Further investigation into the mechanism of action of 25HC may inform novel antiviral therapies and influence the use of mammalian reovirus in clinical trials as an oncolytic agent.

  • A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV [PublishAheadOfPrint]

  • The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks, and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV, and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.

    Importance

    Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation), and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.

  • Geminivirus Replication protein impairs SUMO conjugation of PCNA at two acceptor sites [PublishAheadOfPrint]

  • Geminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (Proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Post-translational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g. replication, recombination, repair, etc.). In yeast, PCNA sumoylation has been associated to DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that co-expression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylated in planta and that Rep also interferes with PCNA sumoylation in plant cells.

    IMPORTANCE

    SUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfering with post-translational modifications of selected host factors to create a proper environment for infection.

  • Real-time visualization and quantification of human Cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology. [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) induces latent life-long infections in all human populations. Between 30 to nearly 100% of individuals are affected depending on geographic area and socio-economic conditions. The biology of this virus is difficult to explore due to its extreme sophistication and the lack of pertinent animal model. Here we present the first application of the ANCHOR DNA labeling system to a herpes virus, enabling real time imaging and direct monitoring of HCMV infection and replication in living human cells. The ANCHOR system is composed of a protein (OR) which specifically binds to a short, non-repetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. When OR protein is fused to GFP, its accumulation results in a site specific fluorescent focus. We have created a recombinant ANCHOR- HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of nearly the complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type specific HCMV behavior and sensitivity to inhibitors. Our results show that the ANCHOR technology provides an efficient tool for the study of complex DNA viruses and a new highly promising system for the development of innovative biotechnology applications.

    IMPORTANCE The ANCHOR technology is to date the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. This technology is applicable to virtually any DNA virus or viruses presenting a dsDNA phase, paving the way to imaging infection in various cell lines or even in animal models, and opening fascinating fundamental and applied prospects. Associated to high content automated microscopy, this technology permitted rapid, robust and precise determination of Ganciclovir IC50 and IC90 on HCMV replication, with minimal hands-on investment. To search for new antiviral activities, the experiment is easy to up-grade towards efficient and cost-effective screening of large chemical libraries. Simple infection of permissive cells with ANCHOR-viruses in the presence of a compound of interest even provides a first estimation of the stage of the viral cycle this molecule is acting upon.

  • A Modified Anoikis Assay That Functionally Segregates LMP1 Strains Into Two Groups [PublishAheadOfPrint]

  • Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein (LMP) 1. During epithelial metastasis, detached cells must overcome anoikis-induced cell death and, gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line, HK1, following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PxQxT motif on LMP1 that activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1 and Med+ promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and non-recovering) and, proposes that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors.

    Importance

    LMP1 is one of the most divergent sequences in the EBV genome that phylogenetically segregate into seven LMP1 strains. LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional mapping studies on LMP1 would support the notion that viral proteins could serve as molecular toolkits, and potentially reveal sequence-associated risk factors in NPC metastatic progression.

  • Gallid Herpesvirus 1 Initiates Apoptosis in Uninfected Cells through Paracrine Repression of p53 [PublishAheadOfPrint]

  • Apoptosis is a common innate defense mechanism of host cells against viral infection and is therefore suppressed by many viruses, including herpes simplex virus (HSV), via various strategies. A recent in vivo study reported the apoptosis of remote uninfected cells during Gallid herpesvirus 1 (GaHV-1) infection, yet little is known about this previously unknown aspect of herpesvirus-host interactions. The aim of the present study was to investigate the apoptosis of uninfected host cells during GaHV-1 infection. The present study used in vitro and in ovo models, which avoided potential interference by host anti-viral immunity, and demonstrated that this GaHV-1-host interaction is independent of host immune responses and important for both the pathological effect of viral infection and early viral dissemination from the primary infection site to distant tissues. Further, we revealed that GaHV-1 infection triggers this process in a paracrine-regulated manner. Using genome-wide transcriptome analyses in combination with a set of functional studies, we found that this paracrine-regulated effect requires the repression of p53 activity in uninfected cells. In contrast, the activation of p53 not only prevented the apoptosis of remote uninfected cells and subsequent pathological damage induced by GaHV-1 infection but also delayed viral dissemination significantly. Moreover, p53 activation repressed viral replication both in vitro and in ovo, suggesting that dual cell-intrinsic mechanisms underlie the suppression of GaHV-1 infection by p53 activation. This study uncovers the mechanism underlying the herpesvirus-triggered apoptosis of remote host cells and extends our understanding of both herpesvirus-host interactions and the roles of p53 in viral infection.

    Importance

    It is well-accepted that herpesviruses suppress the apoptosis of host cells via various strategies to ensure sustained viral replication during infection. However, a recent in vivo study reported the apoptosis of remote uninfected cells during GaHV-1 infection. The mechanism and the biological meaning of this unexpected herpesvirus-host interaction are unclear. This study uncovers the mechanisms of herpesvirus-triggered apoptosis in uninfected cells and may also contribute to a mechanistic illustration of paracrine-regulated apoptosis induced by other viruses in uninfected host cells.

  • The 135 gene of goatpox virus encodes an inhibitor of nuclear factor-{kappa}B and apoptosis and may serve as an improved insertion site to generate vectored live vaccine [PublishAheadOfPrint]

  • Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae. Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an ~18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of nuclear factor-B activation and apoptosis, and is similar to N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene of GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene; a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than that using the traditional tk gene. These results suggest that 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity, and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.

    Importance

    Capripoxviruses are etiological agents of important diseases in sheep, goats and cattle. There are rare reports about viral protein function related to capripoxviruses. Here, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate vectored vaccine resulted in stronger adaptive immune responses than using the tk locus. As capripoxviruses are promising viral-vectored vaccines against many important diseases in small ruminates and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxviruses vectored live dual vaccines.

  • Tomato yellow leaf curl virus V2 interacts with host HDA6 to suppress methylation-mediated transcriptional gene silencing in plants [PublishAheadOfPrint]

  • Cytosine DNA methylation is a conserved epigenetic silencing mechanism that defends against biotic stresses such as geminivirus infection. As a countermeasure, geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Previous studies showed that V2 protein of Tomato yellow leaf curl virus (TYLCV) functions as a TGS suppressor. However, how V2 mediates TGS suppression remains unknown. Here we show that V2 interacts directly with a Nicotiana benthamiana histone deacetylase 6 (NbHDA6), a homolog of Arabidopsis HDA6 (AtHDA6), known to be involved in gene silencing in cooperation with METHYLTRANSFERASE 1 (MET1). NbHDA6 genetically complemented a late-flowering phenotype and restored histone deacetylation of an AtHDA6 mutant. Furthermore, our investigation showed that NbHDA6 displayed histone deacetylase enzymatic activity, which was not inhibited by V2. Genetic analysis revealed that silencing of NbHDA6 expression resulted in enhanced susceptibility to TYLCV infection. In addition, methylation-sensitive PCR and bisulfite sequencing analysis showed that silencing of NbHDA6 expression caused reduced DNA methylation of the viral genome in infected plants. HDA6 was previously shown to recruit and physically interact with MET1 to function in gene silencing. Using competitive pull down and co-immunoprecipitation assays, we demonstrated that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. These findings suggest that V2 interacts with host HDA6 and interferes with the recruitment of MET1 by HDA6 resulting in decreased methylation of the viral DNA genome by TGS with a concomitant increase in host susceptibility to TYLCV infection.

    IMPORTANCE

    Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses. In turn, geminiviruses encode proteins that inhibit methylation by TGS. Previous studied showed that TYLCV V2 can efficiently suppress TGS, but the mechanism remains unknown. We showed that V2 interacted with NbHDA6, but did not inhibit its enzymatic activity. As HDA6 is known to be involved in gene silencing in cooperation with MET1, we explored the relationship between V2, NbMET1 and NbHDA6. Our investigation showed that V2 did not interact, but competed with NbMET1 for direct binding to NbHDA6. To our knowledge, this is the first report that viral proteins inhibit TGS by interacting with histone deacetylase but not by blocking the methyl cycle. This work adds an additional mechanism for TGS suppression by geminiviruses.

  • Equine Myxovirus Resistance Protein 2 Restricts Lentiviral Replication by Blocking Nuclear Uptake of Capsid Protein [PublishAheadOfPrint]

  • Human Myxovirus resistance 2 (huMxB) has been shown to be a determinant type I interferon-induced host factor involved in the inhibition of HIV-1 as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of the integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from non-primate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) on both the equine infectious anemia virus (EIAV) and HIV-1 and investigated possible mechanisms for its specific function. We demonstrated that IFNaalpha;/bbeta; upregulates the expression of eqMx2 in equine monocyte-derived macrophages (eMDMs). Overexpression of eqMx2 significantly suppresses the replication of EIAV, HIV-1, and SIVs, but not that of MLV. Knockdown of eqMx2 transcription weakens the inhibition of EIAV replication by type I interferon. Interestingly, immunofluorescence assays suggest that the subcellular localization of eqMx2 changes following virus infection, from being dispersed in the cytoplasm to being accumulated at the nuclear envelope. Furthermore, eqMx2 blocks the nuclear uptake of the proviral genome by binding to the viral capsid. The N-truncated mutant of eqMx2 lost the ability to bind the viral capsid as well as the restriction effect for lentiviruses. These results improve our understanding of the Mx2 protein in non-primate animals.

    IMPORTANCE Previous research has shown that the antiviral ability of Mx2s is confined to primates, particularly humans. EIAV has been shown to be insensitive to the restriction by human MxB. Here, we described the function of equine Mx2. This protein plays an important role in the suppression of EIAV, HIV-1, and SIVs. The antiviral activity of eqMx2 depends on its subcellular location as well as its capsid binding capacity. Our results showed that following viral infection, eqMx2 changes its original cytoplasmic location and accumulates at the nuclear envelope where it binds to the viral capsid and blocks the nuclear entry of reverse transcribed proviral DNAs. In contrast, huMxB does not bind to the EIAV capsid and shows no EIAV restriction effect. These studies expand our understanding of the function of the equine Mx2 protein.

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