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Table of Contents for this page:

  • Current Issue
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  • Current Issue of Journal of Virology

    JVI Current Issue

  • Reactive Oxygen Species-Mediated c-Jun NH2-Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction [Virus-Cell Interactions]

  • Autophagy is closely associated with the regulation of hepatitis B virus (HBV) replication. HBV X protein (HBx), a multifunctional regulator in HBV-associated biological processes, has been demonstrated to be crucial for autophagy induction by HBV. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we demonstrated that HBx induced autophagosome formation independently of the class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway. In contrast, the class III PI3K(VPS34)/beclin-1 pathway was revealed to be critical for HBx-induced autophagosome formation. Further study showed that HBx did not affect the level of VPS34 and beclin-1 expression but inhibited beclin-1/Bcl-2 association, and c-Jun NH2-terminal kinase (JNK) signaling was found to be important for this process. Moreover, it was found that HBx treatment led to the generation of reactive oxygen species (ROS), and inhibition of ROS activity abrogated both JNK activation and autophagosome formation. Of importance, ROS-JNK signaling was also revealed to play an important role in HBV-induced autophagosome formation and subsequent HBV replication. These data may provide deeper insight into the mechanisms of autophagy induction by HBx and help in the design of new therapeutic strategies against HBV infection.

    IMPORTANCE HBx plays a key role in diverse HBV-associated biological processes, including autophagy induction. However, the molecular mechanisms of autophagy induction by HBx, especially the signaling pathways involved, remain elusive. In the present investigation, we found that HBx induced autophagy independently of the class I PI3K/AKT/mTOR signaling pathway, while the class III PI3K(VPS34)/beclin-1 pathway was revealed to be crucial for this process. Further data showed that ROS-JNK activation by HBx resulted in the release of beclin-1 from its association with Bcl-2 to form a complex with VPS34, thus enhancing autophagosome formation. Of importance, ROS-JNK signaling was also demonstrated to be critical for HBV replication via regulation of autophagy induction. These data help to elucidate the molecular mechanisms of autophagy induction by HBx/HBV and might be useful for designing novel therapeutic approaches to HBV infection.

  • Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity [Genome Replication and Regulation of Viral Gene Expression]

  • Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.

    IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

  • Editorial Board [Masthead]

  • Sensing of HIV-1 Entry Triggers a Type I Interferon Response in Human Primary Macrophages [Pathogenesis and Immunity]

  • Along with CD4+ T lymphocytes, macrophages are a major cellular source of HIV-1 replication and a potential viral reservoir. Following entry and reverse transcription in macrophages, cloaking of the viral cDNA by the HIV-1 capsid limits its cytosolic detection, enabling efficient replication. However, whether incoming HIV-1 particles are sensed by macrophages prior to reverse transcription remains unclear. Here, we show that HIV-1 triggers a broad expression of interferon (IFN)-stimulated genes (ISG) in monocyte-derived macrophages within a few hours after infection. This response does not require viral reverse transcription or the presence of HIV-1 RNA within particles, but viral fusion is essential. This response is elicited by viruses carrying different envelope proteins and thus different receptors to proceed for viral entry. Expression of ISG in response to viral entry requires TBK1 activity and type I IFNs signaling. Remarkably, the ISG response is transient but affects subsequent viral spread. Together, our results shed light on an early step of HIV-1 sensing by macrophages at the level of entry, which confers an early protection through type I IFN signaling and has potential implications in controlling the infection.

    IMPORTANCE HIV infection is restricted to T lymphocytes and macrophages. HIV-1-infected macrophages are found in many tissues of infected patients, even under antiretroviral therapy, and are considered a viral reservoir. How HIV-1 is detected and what type of responses are elicited upon sensing remain in great part elusive. The kinetics and localization of the production of cytokines such as interferons in response to HIV is of critical importance to understanding how the infection and the immune response are established. Our study provides evidence that macrophages can detect HIV-1 as soon as it enters the cell. Interestingly, this sensing is independent of the presence of viral nucleic acids within the particles but requires their fusion with the macrophages. This triggers a low interferon response, which activates an antiviral program protecting cells against further viral challenge and thus potentially limiting the spread of the infection.

  • Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States [Vaccines and Antiviral Agents]

  • The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Env-activating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0ddeg;C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptor-binding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.

    IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0ddeg;C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sensitivity by chemical agents that affect the structural stability of proteins can be applied to increase the potency of topical microbicides and vaccine-elicited antibodies.

  • Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab [Vaccines and Antiviral Agents]

  • Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site OOslash; at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro. In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site OOslash; are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site OOslash;-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.

    IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, high-potency neutralizing antibody (5C4) that recognizes antigenic site OOslash; and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the mechanisms of neutralization has the potential to identify improved products for the prevention and treatment of RSV infection.

  • Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate [Vaccines and Antiviral Agents]

  • Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation.

    IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site OOslash; binding and greater protection against RSV challenge.

  • Contribution of Human Lung Parenchyma and Leukocyte Influx to Oxidative Stress and Immune System-Mediated Pathology following Nipah Virus Infection [Pathogenesis and Immunity]

  • Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD-SCID/ mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the bone marrow, liver, and thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections. Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system-reconstituted mice in order to identify the overall effect of immune cells on NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in lung grafts. In addition, the presence of human leukocytes increased the expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI and inform efforts to identify therapeutic targets.

    IMPORTANCE Nipah virus (NiV) is an emerging paramyxovirus that can cause a lethal respiratory and neurological disease in humans. Only limited data are available on NiV pathogenesis in the human lung, and the relative contribution of the innate immune response and NiV to acute lung injury (ALI) is still unknown. Using human lung grafts in a human immune system-reconstituted mouse model, we showed that the NiV Bangladesh strain induced cytopathic lesions in lung grafts similar to those described in patients irrespective of the donor origin or the presence of leukocytes. However, the human immune system interfered with virus spread, responded to infection by leukocyte infiltration in the small airways and alveolar area, induced oxidative stress, and triggered the production of cytokines and chemokines that regulate inflammatory influx by leukocytes in response to infection. Understanding how leukocytes interact with NiV and cause ALI in human lung xenografts is crucial for identifying therapeutic targets.

  • Molluscum Contagiosum Virus MC159 Abrogates cIAP1-NEMO Interactions and Inhibits NEMO Polyubiquitination [Virus-Cell Interactions]

  • Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-B activation, a powerful antiviral response, via interactions with the NF-B essential modulator (NEMO) subunit of the IB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-B activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-B activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus.

    IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-B activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-B. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells.

  • Chikungunya Virus Overcomes Polyamine Depletion by Mutation of nsP1 and the Opal Stop Codon To Confer Enhanced Replication and Fitness [Vaccines and Antiviral Agents]

  • Polyamines, which are small positively charge molecules present in all cells, play important roles in the replication of DNA and RNA viruses. Chikungunya virus (CHIKV) relies on polyamines for translation of the viral genome upon viral entry, and pharmacological depletion of polyamines limits viral replication. However, the potential development of antiviral resistance necessitates a better understanding of how polyamines function and can be targeted via compounds that alter polyamine levels. We have isolated CHIKV that is resistant to polyamine depletion and contains two mutations in the nonstructural protein 1 (nsP1)-coding region in combination with a mutation to the opal stop codon preceding nsP4. These mutations, in addition to promoting viral replication in polyamine-depleted cells, confer enhanced viral replication in vitro and in vivo. The nsP1 mutations enhance membrane binding and methyltransferase activities, while the stop codon mutation allows increased downstream translation. These mutations, when combined, enhance viral fitness, but individual mutants are attenuated in mosquitoes. Together, our results suggest that CHIKV can evolve resistance to polyamine depletion and that pharmaceuticals targeting the polyamine biosynthetic pathway may be best used in combination with other established antivirals to mitigate the development of resistance.

    IMPORTANCE Chikungunya virus is a mosquito-borne virus that has infected millions worldwide. Its expansion into the Americas and rapid adaptation to new mosquito hosts present a serious threat to human health, which we can combat with the development of antiviral therapies as well as understanding how these viruses will mutate when exposed to antiviral therapies. Targeting polyamines, small positively charged molecules in the cell, may be a potential strategy against RNA viruses, including chikungunya virus. Here, we have described a virus that is resistant to polyamine depletion and has increased fitness in cells and in full organisms. Mutations in viral genome capping machinery, membrane binding activity, and a stop codon arise, and their altered activities enhance replication in the absence of polyamines. These results highlight strategies by which chikungunya virus can overcome polyamine depletion and emphasize continued research on developing improved antiviral therapies.

  • Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1 [Genome Replication and Regulation of Viral Gene Expression]

  • Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate.

    IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.

  • Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses [Virus-Cell Interactions]

  • Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts.

    IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.

  • Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling [Virus-Cell Interactions]

  • Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.

    IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

  • Natural Variation of Epstein-Barr Virus Genes, Proteins, and Primary MicroRNA [Genetic Diversity and Evolution]

  • Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains, including many primary isolates, have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1, and the BART microRNA (miRNA) cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains, named QCIGP, results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through single-nucleotide polymorphisms (SNPs) in the primary miRNA outside the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future, more directed analysis of association of specific EBV variations with EBV biology and EBV-associated diseases.

    IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Thus, relationships between EBV genome sequence variation and health, disease, geography, and ethnicity of the host may be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focusing on variation in LMP1, Zp, gp350, EBNA1, and the BART miRNA cluster 2, new relationships with the known type 1/type 2 strains are demonstrated, and a novel classification of EBNA1 and the BART miRNAs is proposed.

  • Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture [Virus-Cell Interactions]

  • Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus.

    IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

  • Differential Disease Susceptibilities in Experimentally Reptarenavirus-Infected Boa Constrictors and Ball Pythons [Pathogenesis and Immunity]

  • Inclusion body disease (IBD) is an infectious disease originally described in captive snakes. It has traditionally been diagnosed by the presence of large eosinophilic cytoplasmic inclusions and is associated with neurological, gastrointestinal, and lymphoproliferative disorders. Previously, we identified and established a culture system for a novel lineage of arenaviruses isolated from boa constrictors diagnosed with IBD. Although ample circumstantial evidence suggested that these viruses, now known as reptarenaviruses, cause IBD, there has been no formal demonstration of disease causality since their discovery. We therefore conducted a long-term challenge experiment to test the hypothesis that reptarenaviruses cause IBD. We infected boa constrictors and ball pythons by cardiac injection of purified virus. We monitored the progression of viral growth in tissues, blood, and environmental samples. Infection produced dramatically different disease outcomes in snakes of the two species. Ball pythons infected with Golden Gate virus (GoGV) and with another reptarenavirus displayed severe neurological signs within 2 months, and viral replication was detected only in central nervous system tissues. In contrast, GoGV-infected boa constrictors remained free of clinical signs for 2 years, despite high viral loads and the accumulation of large intracellular inclusions in multiple tissues, including the brain. Inflammation was associated with infection in ball pythons but not in boa constrictors. Thus, reptarenavirus infection produces inclusions and inclusion body disease, although inclusions per se are neither necessarily associated with nor required for disease. Although the natural distribution of reptarenaviruses has yet to be described, the different outcomes of infection may reflect differences in geographical origin.

    IMPORTANCE New DNA sequencing technologies have made it easier than ever to identify the sequences of microorganisms in diseased tissues, i.e., to identify organisms that appear to cause disease, but to be certain that a candidate pathogen actually causes disease, it is necessary to provide additional evidence of causality. We have done this to demonstrate that reptarenaviruses cause inclusion body disease (IBD), a serious transmissible disease of snakes. We infected boa constrictors and ball pythons with purified reptarenavirus. Ball pythons fell ill within 2 months of infection and displayed signs of neurological disease typical of IBD. In contrast, boa constrictors remained healthy over 2 years, despite high levels of virus throughout their bodies. This difference matches previous reports that pythons are more susceptible to IBD than boas and could reflect the possibility that boas are natural hosts of these viruses in the wild.

  • Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles [Structure and Assembly]

  • Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.

    IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

  • Infectious Prions in the Pregnancy Microenvironment of Chronic Wasting Disease-Infected Reeves' Muntjac Deer [Prions]

  • Ample evidence exists for the presence of infectious agents at the maternal-fetal interface, often with grave outcomes to the developing fetus (i.e., Zika virus, brucella, cytomegalovirus, and toxoplasma). While less studied, pregnancy-related transmissible spongiform encephalopathies (TSEs) have been implicated in several species, including humans. Our previous work has shown that prions can be transferred from mother to offspring, resulting in the development of clinical TSE disease in offspring born to muntjac dams infected with chronic wasting disease (CWD) (1). We further demonstrated protein misfolding cyclic amplification (PMCA)-competent prions within the female reproductive tract and in fetal tissues harvested from CWD experimentally and naturally exposed cervids (1, 2). To assess whether the PMCA-competent prions residing at the maternal-fetal interface were infectious and to determine if the real-time quaking-induced conversion (RT-QuIC) methodology may enhance our ability to detect amyloid fibrils within the pregnancy microenvironment, we employed a mouse bioassay and RT-QuIC. In this study, we have demonstrated RT-QuIC seeding activity in uterus, placentome, ovary, and amniotic fluid but not in allantoic fluids harvested from CWD-infected Reeves' muntjac dams showing clinical signs of infection (clinically CWD-infected) and in some placentomes from pre-clinically CWD-infected dams. Prion infectivity was confirmed within the uterus, amniotic fluid, and the placentome, the semipermeable interface that sustains the developing fetus, of CWD-infected dams. This is the first report of prion infectivity within the cervid pregnancy microenvironment, revealing a source of fetal CWD exposure prior to the birthing process, maternal grooming, or encounters with contaminated environments.

    IMPORTANCE The facile dissemination of chronic wasting disease within captive and free-range cervid populations has led to questions regarding the transmission dynamics of this disease. Direct contact with infected animals and indirect contact with infectious prions in bodily fluids and contaminated environments are suspected to explain the majority of this transmission. A third mode of transmission, from mother to offspring, may be underappreciated. The presence of pregnancy-related prion infectivity within the uterus, amniotic fluid, and the placental structure reveals that the developing fetus is exposed to a source of prions long before exposure to the infectious agent during and after the birthing process or via contact with contaminated environments. These findings have impact on our current concept of CWD disease transmission.

  • Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses [Virus-Cell Interactions]

  • Akabane virus (AKAV) and Schmallenberg virus (SBV) are members of the genus Orthobunyavirus, which are transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo. Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro, to promote virus replication in susceptible cells.

    IMPORTANCE AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.

  • A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model [Virus-Cell Interactions]

  • Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice.

    IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

  • Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA [Virus-Cell Interactions]

  • Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ~5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.

    IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.

  • Deletion of the K1L Gene Results in a Vaccinia Virus That Is Less Pathogenic Due to Muted Innate Immune Responses, yet Still Elicits Protective Immunity [Pathogenesis and Immunity]

  • All viruses strategically alter the antiviral immune response to their benefit. The vaccinia virus (VACV) K1 protein has multiple immunomodulatory effects in tissue culture models of infection, including NF-B antagonism. However, the effect of K1 during animal infection is poorly understood. We determined that a K1L-less vaccinia virus (vK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity was correlated with diminished virus replication in intranasally infected mice. However, in intradermally inoculated ears, vK1L replicated to levels nearly identical to those of VACV, implying that the decreased immune response to vK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vK1L induced slightly less edema than vK1L, as revealed by histopathology and noninvasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vK1L-infected ears. While these results identified the biological basis for smaller lesions, they remained puzzling; because K1 antagonizes NF-B in vitro, antiviral gene expression was expected to be higher during vK1L infection. Despite these diminished innate immune responses, vK1L vaccination induced a protective VACV-specific CD8+ T cell response and protected against a lethal VACV challenge. Thus, vK1L is the first vaccinia virus construct reported that caused a muted innate immune gene expression profile and decreased immune cell infiltration in an intradermal model of infection yet still elicited protective immunity.

    IMPORTANCE The vaccinia virus (VACV) K1 protein inhibits NF-B activation among its other antagonistic functions. A virus lacking K1 (vK1L) was predicted to be less pathogenic because it would trigger a more robust antiviral immune response than VACV. Indeed, vK1L was less pathogenic in intradermally infected mouse ear pinnae. However, vK1L infection unexpectedly elicited dramatically reduced infiltration of innate immune cells into ears. This was likely due to decreased expression of cytokine and chemokine genes in vK1L-infected ears. As such, our finding contradicted observations from cell culture systems. Interestingly, vK1L conferred protective immunity against lethal VACV challenge. This suggests that the muted immune response triggered during vK1L infection remained sufficient to mount an effective protective response. Our results highlight the complexity and unpredictable nature of virus-host interactions, a relationship that must be understood to better comprehend virus pathogenesis or to manipulate viruses for use as vaccines.

  • Molluscum Contagiosum Virus Protein MC005 Inhibits NF-{kappa}B Activation by Targeting NEMO-Regulated I{kappa}B Kinase Activation [Virus-Cell Interactions]

  • Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-B activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-B drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-B activation. MC005 inhibited NF-B proximal to the IB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-B essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions.

    IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-B, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-B by MC005 confirms current models of the complex way in which NF-B is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.

  • Development of Clade-Specific and Broadly Reactive Live Attenuated Influenza Virus Vaccines against Rapidly Evolving H5 Subtype Viruses [Vaccines and Antiviral Agents]

  • We have developed pandemic live attenuated influenza vaccines (pLAIVs) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted (ca) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (clade of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt, which prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs. The first approach was to modify the hemagglutinin gene of a highly pathogenic wild-type (wt) clade virus, A/Egypt/N03434/2009 (Egy/09) (H5N1), with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca. The second approach was to select a low-pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses, and contained a novel NA subtype for humans. We selected the low-pathogenicity A/duck/Hokkaido/69/2000 (H5N3) (dk/Hok/00) virus for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific for the gs/GD lineage viruses but did not neutralize recent North American isolates (clade, whereas antisera from dk/Hok/69 ca-vaccinated ferrets cross-reacted with clade and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses.

    IMPORTANCE In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed by using two different approaches: one targeted a specific circulating strain in Egypt, and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches, and the data support the further development of both vaccine viruses to optimally prepare for the further spread of clade 2.2.1 or viruses.

  • Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins [Structure and Assembly]

  • The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vI2. As anticipated, vI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes.

    IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry.

  • Uncovering the Repertoire of Endogenous Flaviviral Elements in Aedes Mosquito Genomes [Genetic Diversity and Evolution]

  • Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.

    IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system.

  • Roles of M1 and M2 Macrophages in Herpes Simplex Virus 1 Infectivity [Pathogenesis and Immunity]

  • Macrophages are the predominant infiltrate in the corneas of mice that have been ocularly infected with herpes simplex virus 1 (HSV-1). However, very little is known about the relative roles of M1 (classically activated or polarized) and M2 (alternatively activated or polarized) macrophages in ocular HSV-1 infection. To better understand these relationships, we assessed the impact of directed M1 or M2 activation of RAW264.7 macrophages and peritoneal macrophages (PM) on subsequent HSV-1 infection. In both the RAW264.7 macrophage and PM in vitro models, HSV-1 replication in M1 macrophages was markedly lower than in M2 macrophages and unstimulated controls. The M1 macrophages expressed significantly higher levels of 28 of the 32 tested cytokines and chemokines than M2 macrophages, with HSV-1 infection significantly increasing the levels of proinflammatory cytokines and chemokines in the M1 versus the M2 macrophages. To examine the effects of shifting the immune response toward either M1 or M2 macrophages in vivo, wild-type mice were injected with gamma interferon (IFN-) DNA or colony-stimulating factor 1 (CSF-1) DNA prior to ocular infection with HSV-1. Virus replication in the eye, latency in trigeminal ganglia (TG), and markers of T cell exhaustion in the TG were determined. We found that injection of mice with IFN- DNA, which enhances the development of M1 macrophages, increased virus replication in the eye; increased latency; and also increased CD4, CD8, IFN-, and PD-1 transcripts in the TG of latently infected mice. Conversely, injection of mice with CSF-1 DNA, which enhances the development of M2 macrophages, was associated with reduced virus replication in the eye and reduced latency and reduced the levels of CD4, CD8, IFN-,and PD-1 transcripts in the TG. Collectively, these results suggest that M2 macrophages directly reduce the levels of HSV-1 latency and, thus, T-cell exhaustion in the TG of ocularly infected mice.

    IMPORTANCE Our findings demonstrate a novel approach to further reducing HSV-1 replication in the eye and latency in the TG by modulating immune components, specifically, by altering the phenotype of macrophages. We suggest that inclusion of CSF-1 as part of any vaccination regimen against HSV infection to coax responses of macrophages toward an M2, rather than an M1, response may further improve vaccine efficacy against ocular HSV-1 replication and latency.

  • Anti-HIV-1 ADCC Antibodies following Latency Reversal and Treatment Interruption [Pathogenesis and Immunity]

  • There is growing interest in utilizing antibody-dependent cellular cytotoxicity (ADCC) to eliminate infected cells following reactivation from HIV-1 latency. A potential barrier is that HIV-1-specific ADCC antibodies decline in patients on long-term antiretroviral therapy (ART) and may not be sufficient to eliminate reactivated latently infected cells. It is not known whether reactivation from latency with latency-reversing agents (LRAs) could provide sufficient antigenic stimulus to boost HIV-1-specific ADCC. We found that treatment with the LRA panobinostat or a short analytical treatment interruption (ATI), 21 to 59 days, was not sufficient to stimulate an increase in ADCC-competent antibodies, despite viral rebound in all subjects who underwent the short ATI. In contrast, a longer ATI, 2 to 12 months, among subjects enrolled in the Strategies for Management of Antiretroviral Therapy (SMART) trial robustly boosted HIV-1 gp120-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a lag between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies toward eliminating latently infected cells.

    IMPORTANCE The "shock and kill" HIV-1 cure strategy aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. Several latency reversing agents (LRAs) have been examined in vivo, but LRAs alone have not been able to achieve HIV-1 remission and prevent viral rebound following analytical treatment interruption (ATI). In this study, we examined whether LRA treatment or ATI can provide sufficient antigenic stimulus to boost HIV-1-specific functional antibodies that can eliminate HIV-1-infected cells. Our study has implications for the antigenic stimulus required for antilatency strategies and/or therapeutic vaccines to boost functional antibodies and assist in eliminating the latent reservoir.

  • Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-B signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-B and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-B and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

  • Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2 [Virus-Cell Interactions]

  • The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvB1) produced using a complementing cell line expressing the viral kinase. Examination of vvB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.

    IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.

  • Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation [Virus-Cell Interactions]

  • Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus.

    IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease.

  • The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores [Structure and Assembly]

  • The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.

  • A Novel A(H7N2) Influenza Virus Isolated from a Veterinarian Caring for Cats in a New York City Animal Shelter Causes Mild Disease and Transmits Poorly in the Ferret Model [Pathogenesis and Immunity]

  • In December 2016, a low-pathogenic avian influenza (LPAI) A(H7N2) virus was identified to be the causative source of an outbreak in a cat shelter in New York City, which subsequently spread to multiple shelters in the states of New York and Pennsylvania. One person with occupational exposure to infected cats became infected with the virus, representing the first LPAI H7N2 virus infection in a human in North America since 2003. Considering the close contact that frequently occurs between companion animals and humans, it was critical to assess the relative risk of this novel virus to public health. The virus isolated from the human case, A/New York/108/2016 (NY/108), caused mild and transient illness in ferrets and mice but did not transmit to naive cohoused ferrets following traditional or aerosol-based inoculation methods. The environmental persistence of NY/108 virus was generally comparable to that of other LPAI H7N2 viruses. However, NY/108 virus replicated in human bronchial epithelial cells with an increased efficiency compared with that of previously isolated H7N2 viruses. Furthermore, the novel H7N2 virus was found to utilize a relatively lower pH for hemagglutinin activation, similar to human influenza viruses. Our data suggest that the LPAI H7N2 virus requires further adaptation before representing a substantial threat to public health. However, the reemergence of an LPAI H7N2 virus in the northeastern United States underscores the need for continuous surveillance of emerging zoonotic influenza viruses inclusive of mammalian species, such as domestic felines, that are not commonly considered intermediate hosts for avian influenza viruses.

    IMPORTANCE Avian influenza viruses are capable of crossing the species barrier to infect mammals, an event of public health concern due to the potential acquisition of a pandemic phenotype. In December 2016, an H7N2 virus caused an outbreak in cats in multiple animal shelters in New York State. This was the first detection of this virus in the northeastern United States in over a decade and the first documented infection of a felid with an H7N2 virus. A veterinarian became infected following occupational exposure to H7N2 virus-infected cats, necessitating the evaluation of this virus for its capacity to cause disease in mammals. While the H7N2 virus was associated with mild illness in mice and ferrets and did not spread well between ferrets, it nonetheless possessed several markers of virulence for mammals. These data highlight the promiscuity of influenza viruses and the need for diligent surveillance across multiple species to quickly identify an emerging strain with pandemic potential.

  • Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers [Vaccines and Antiviral Agents]

  • Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged.

    IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

  • Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion [Genetic Diversity and Evolution]

  • The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.

    IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.

  • Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection [Pathogenesis and Immunity]

  • Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (ggt;2-month) papillomas (~20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (~15%); and (iii) no visible papillomas and viral clearance (~65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.

    IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

  • Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes [Genome Replication and Regulation of Viral Gene Expression]

  • Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.

    IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.

  • A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junin Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR) [Virus-Cell Interactions]

  • Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Juniiacute;n virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2aalpha;. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2aalpha;, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2aalpha;, even following exposure to the synthetic dsRNA poly(Immiddot;C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2aalpha; phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.

    IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Juniiacute;n virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

  • Classic Spotlight, 1996 and 1997: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

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

  • Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E [Virus-Cell Interactions]

  • Interactions between hepatitis C virus (HCV) and lipoproteins in humans play an important role in the efficient establishment of chronic infection. Apolipoprotein E (ApoE) on the HCV envelope mediates virus attachment to host cells as well as immune evasion. This interaction is thought to occur in hepatocytes, as ApoE plays dual functions in HCV assembly and maturation as well as cell attachment. In the present study, we found that secreted ApoE (sApoE) can also bind to viral particles via its C-terminal domain after HCV is released from the cell. Furthermore, the binding affinity of interactions between the sApoE N terminus and cell surface receptors affected HCV infectivity in a dose-dependent manner. The extracellular binding of sApoE to HCV is dependent on HCV envelope proteins, and recombinant HCV envelope proteins are also able to bind to sApoE. These results suggest that extracellular interactions between HCV and sApoE may potentially complicate vaccine development and studies of viral pathogenesis.

    IMPORTANCE End-stage liver disease caused by chronic HCV infection remains a clinical challenge, and there is an urgent need for a prophylactic method of controlling HCV infection. Because host immunity against HCV is poorly understood, additional investigations of host-virus interactions in the context of HCV are important. HCV is primarily transmitted through blood, which is rich in lipoproteins. Therefore, it is of interest to further determine how HCV interacts with lipoproteins in human blood. In this study, we found that secreted ApoE (sApoE), an exchangeable component found in lipoproteins, participates in extracellular interactions with HCV virions. More significantly, different variants of sApoE differentially affect HCV infection efficiency in a dose-dependent manner. These findings provide greater insight into HCV infection and host immunity and could help propel the development of new strategies for preventing HCV infection.

  • Dok-1 and Dok-2 Are Required To Maintain Herpes Simplex Virus 1-Specific CD8+ T Cells in a Murine Model of Ocular Infection [Pathogenesis and Immunity]

  • Dok-1 and Dok-2 negatively regulate responses downstream of several immune receptors in lymphoid and myeloid cells. Recent evidence showed that Dok proteins are essential in the formation of memory CD8+ T cells to an exogenous epitope expressed by vaccinia virus; however, the importance of Dok-1 and Dok-2 in the control of viral infection is unknown. Here, we investigated the role of Dok proteins in modulating the immune response against herpes simplex virus 1 (HSV-1) in a mouse model of ocular infection. During acute infection, viral titers in the eye were similar in wild-type (WT) and Dok-1 and Dok-2 double-knockout (DKO) mice, and the percentages of infiltrating leukocytes were similar in DKO and WT corneas and trigeminal ganglia (TG). DKO mice exhibited a diminished CD8+ T cell response to the immunodominant HSV-1 glycoprotein B (gB) epitope in the spleen and draining lymph nodes compared to WT mice during acute infection. Remarkably, gB-specific CD8+ T cells almost completely disappeared in the spleens of DKO mice during latency, and the reduction of CD8+ effector memory T (Tem) cells was more severe than that of CD8+ central memory T (Tcm) cells. The percentage of gB-specific CD8+ T cells in TG during latency was also dramatically reduced in DKO mice; however, they were phenotypically similar to those from WT mice. In ex vivo assays, reactivation was detected earlier in TG cultures from infected DKO versus WT mice. Thus, Dok-1 and Dok-2 promote survival of gB-specific CD8+ T cells in TG latently infected with HSV-1.

    IMPORTANCE HSV-1 establishes lifelong latency in sensory neurons of trigeminal ganglia (TG). In humans, HSV-1 is able to sporadically reactivate from latently infected neurons and establish a lytic infection at a site to which the neurons project. Most herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. CD8+ T cells are thought to play an important role in controlling recurrent infections. In this study, we examined the involvement of Dok-1 and Dok-2 signaling proteins in the control of HSV-1 infection. We provide evidence that Dok proteins are required to maintain a CD8+ T cell response against HSV-1 during latencymmdash;especially CD8+ Tem cellsmmdash;and that they negatively affect HSV-1 reactivation from latency. Elucidating Dok-mediated mechanisms involved in the control of HSV-1 reactivation from latency might contribute to the development of therapeutic strategies to prevent recurrent HSV-1-induced pathology.

  • Congenital Human Cytomegalovirus Infection and the Enigma of Maternal Immunity [Gem]

  • Human cytomegalovirus (HCMV) is the most common viral infection acquired by the developing human fetus and can result in damage to the developing central nervous system. Although vaccine development to modify this congenital infection is ongoing, the unique epidemiology of maternal HCMV infections appears discordant with strategies for vaccine development. Several characteristics of congenital HCMV infections suggest that the efficacy of vaccines designed to induce responses similar to those that follow natural infection will be limited.

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    JVI Accepts: Articles Published Ahead of Print

  • Intracellular Colocalization of Influenza Viral RNA and Rab11A is Dependent upon Microtubule Filaments [PublishAheadOfPrint]

  • Influenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where sub-complexes of vRNA export from the nucleus and assemble en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments transport via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A-RE primarily transport along MT; therefore, investigation into the role of MT on vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all cell types was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A-RE independent mechanism.

    Importance IAV infections cause large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAV emerge through reassortment of vRNA in animal or human hosts. Elucidating the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly extend previous studies characterizing vRNA assembly. This study is the first to assess the role of MT on influenza replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT to facilitate association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may transport through the cytoplasm without Rab11A-RE in the absence of intact MT. These results enhance our current understanding of vRNA assembly and the role of cytoskeletal proteins in that process.

  • UV Radiation Activates Toll-like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus Type 38 E6 and E7 Oncoproteins [PublishAheadOfPrint]

  • Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergise with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes (PHKs) as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist Np73aalpha;, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we have shown that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.

    IMPORTANCE Beta HPV types have been suggested to act as co-factors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We have shown that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in PHKs. Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.

  • Rab5 and Rab11 are required for clathrin-dependent endocytosis of Japanese encephalitis virus in BHK-21 cells [PublishAheadOfPrint]

  • During infection Japanese Encephalitis Virus generally enters host cells via receptor-mediated clathrin dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and post-internalization of JEV were analyzed using biochemical inhibitors, RNA interference and dominant negative mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol, but not caveolae or macropinocytosis. The effect of dominant-negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined on JEV infection. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 minutes after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.

    IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and post-internalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. This is the first report to show that entry of JEV into BHK-21 cells requires a low pH environment, the process is dynamin-, actin-, and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

  • Synthetic TLR4 and TLR7 ligands work additively via MyD88 to induce protective antiviral immunity in mice [PublishAheadOfPrint]

  • We have previously demonstrated that the combination of synthetic small molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity protective against influenza viruses in homologous, heterologous and heterosubtypic murine challenge models. Combining TLR4 and TLR7 ligands balances Th1/Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors with negligible off-target effects to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow and non-bone marrow derived, radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunizing with this adjuvant and recombinant hemagglutinin antigen could be transferred with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice did not impair protection from a homologous challenge; however, it did significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allowed for significant dose reduction of each component to achieve a level of protection equivalent to that afforded by either single agent at full dose.

    Importance Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen to induce humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

  • NKp46 recognizes the sigma1 protein of reovirus: Implications for reovirus-based cancer therapy [PublishAheadOfPrint]

  • The recent approval of onclolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK activating receptor, NKp46, and its mouse orthologous protein, NCR1, recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify NKp46/NCR1 binding sites to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro. Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo. Collectively, we identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed for both clearance of reovirus infections and reovirus-based tumor therapy.

    Importance Reovirus infects much of the population during childhood, causing mild disease, hence considered being efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We describe here that Natural Killer (NK) cells recognize a viral protein named sigma1 through the NK killer receptor NKp46. Using several mouse tumor models we demonstrate the importance of NK cells in protection from reovirus infection and in reovireus-killing of tumors in vivo. Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in controlling reovirus infections and in reovirus-based cancer therapy.

  • Stability of Cucumber Necrosis Virus at the Quasi Six-fold Axis Affects Zoospore Transmission. [PublishAheadOfPrint]

  • Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus, Olpidium bornovanus. As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (1). P73 lies immediately adjacent to a putative zinc binding site (2) that is formed by three icosahedrally related His residues in the N-termini of the C subunit at the quasi 6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild type CNV in the native and swollen state and the transmission defective mutant, P73G, under native conditions. With the wild type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi 6-fold at the bbeta;-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the bbeta;-annulus had been disrupted and this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the bbeta;-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.

    Importance: Cucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus, Olpidium bornovanus. While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (2), blocks the virus from zoospore transmission while not significantly affecting replication in plants (2). Here we show that the P73G mutant is less stable than wild type and this appears to be correlated with destabilization of the bbeta;-annulus at the icosahedral 3-fold axes. Therefore, the bbeta;-annulus appears to not be essential for particle assembly but is necessary for interactions with the transmission vector.

  • A unique T cell receptor amino acid sequence selected by HTLV-I Tax301-309-specific cytotoxic T-cells in HLA-A24:02+ asymptomatic carriers and adult T-cell leukemia/lymphoma patients [PublishAheadOfPrint]

  • We previously reported that the T-cell receptor (TCR) repertoire of HTLV-I Tax301-309-specific CD8+ cytotoxic T-cells (Tax-CTLs) was highly restricted and a particular amino acid sequence motif, "PDR", was conserved among HLA-A*24:02+ ATL patients who have undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-I. On the other hand, the TCR repertoires in Tax-CTL of asymptomatic HTLV-I carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity determining region 3 (CDR3) of TCR-bbeta; chain of Tax301-309-CTLs at the single-cell level and compared not only TCR repertoires but also the frequencies and phenotypes of Tax-CTL between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax-CTLs, 1458 Tax-CTLs and 140 clones were identified in this cohort. Tax-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and "PDR", the unique motif in TCR-bbeta; CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+CTL frequencies and HTLV-I proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, "PDR", as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-I-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL.

    IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-I virus infection. The HTLV-I virus regulatory protein Tax aggressively promotes the proliferation of HTLV-I-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax-CTL, we found that a unique amino acid sequence motif, "PDR", in CDR3 of TCR-bbeta; chain of Tax-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+Tax-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-I. On the other hand, it remains unclear how Tax-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and "PDR", the unique motif in TCR-bbeta; CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection.

  • STLV-1 infection of Papio Anubis: tax Sequence Heterogeneity and T Cell Recognition [PublishAheadOfPrint]

  • Natural infection of baboons with simian T lymphotropic virus (STLV) is a potentially useful model system for study of vaccination against the human virus HTLV. Here we expand the number of available full-length STLV-1 baboon sequences from one to three and relate T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these lines revealed them to be closely related but distinct from each other and from the STLV-1 baboon sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more IFN- and TNF-aalpha; in response to Tax peptides corresponding to their own sequence than with Tax peptides corresponding to the reference baboon sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed only in tax sequences obtained from PBMC at 7% in 12141 and 56% in 12752.

    Importance: It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventative vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) being naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what is seen with HTLV infection of humans. These results justify the use of STLV-baboon model systems for vaccine development efforts.

  • Binding of HSV-1 UL20 to GODZ affects its palmitoylation and is essential for infectivity and proper targeting and localization of UL20 and gK [PublishAheadOfPrint]

  • HSV-1 UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a non-glycosylated envelope protein that is regulated as a 1 gene. Two-hybrid and pull-down assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (a.k.a.: DHHC3), a cellular Golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication in vitro and affected the localization of UL20 and gK and their interactions but not gC. GODZ is involved in palmitoylation and we found that UL20 is palmitoylated by GODZ using GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK, but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity.

    IMPORTANCE HSV-1 UL20 is a non-glycosylated essential envelope protein that is highly conserved among herpes viruses. In this study, we have shown that: (1) HSV-1 UL20 binds to GODZ (aka: DHHC3), a Golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein; (2) A GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and gK; and 3) UL20 is palmitoylated by GODZ and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered as a potential alternative therapy in not only HSV-1, but other conditions in which GODZ processing is an integral component of pathogenesis.

  • Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity Dependent and Independent Mechanisms [PublishAheadOfPrint]

  • Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so far unknown mechanism, suggesting that CH25H might exert antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by siRNA promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by co-immunoprecipitation and immunofluorescence co-localization analyses, confirmed that both CH25H and CH25H-M interact with the non-structural protein 1 alpha (nsp1aalpha;) of PRRSV. Unexpectedly, the expression of nsp1aalpha; decreased following co-expression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1aalpha; through the ubiquitin--proteasome pathway and that site K169 in the nsp1aalpha; protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration, as well as degrading nsp1aalpha;, revealing a novel antiviral mechanism used by CH25H.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously been a threat to the global swine industry and current vaccines are insufficient to provide sustainable control. Cholesterol 25-hydroxylase (CH25H) has been found to exert a broad antiviral effect, and thus is an attractive target for the development of anti-PRRSV drugs. Herein, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration, but also by degrading viral protein through the ubiquitin--proteasome pathway, suggesting that CH25H is a potential candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.

  • rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming [PublishAheadOfPrint]

  • Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant-priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaMnndash;mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated in SAR-induced plants via induction of salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of salicylic acid analogue BTH and Ca2+ ionophore, A23187, revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceives both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acidnndash;mediated antiviral responses.

    IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaMnndash;mediated SAR illustrates the growth vs. defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

  • Species specificity of vaccinia virus complement control protein towards bovine classical pathway is governed primarily by direct interaction of its acidic residues with factor I [PublishAheadOfPrint]

  • Poxviruses display species tropism nndash; variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE exhibit selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP display selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species-specificity of VCP and SPICE towards the classical pathway (CP). We observed that VCP is ~43-fold superior in inhibiting bovine CP than SPICE. Further, functional assays revealed that increased inhibitory activity of VCP towards bovine CP is solely due to its enhanced cofactor activity with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino acid substitution mutants wherein one or more of the 11 variant VCP residues were substituted onto the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120 and E144 are majorly responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I, but not bovine C4(H2O) (a conformationally similar moiety to C4b). Mapping of these residues onto the modelled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals.

    IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. The reason for this tropism however is unknown. It is well recognized that the virus is susceptible to neutralization by the classical complement pathway (CP). Because the virus encodes a soluble complement regulator VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP, and this is primarily determined by its amino acids E108, E120 and E144, which interact with bovine serine protease factor I to inactivate bovine C4b nndash; one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby fitness of the virus in cattle.

  • ISG15 modulates type-I interferon signaling and antiviral response during hepatitis E virus replication [PublishAheadOfPrint]

  • Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes a self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals especially in solid organ transplant recipients. Innate immunity, via type-I interferon (IFN) response, plays an important role during initial stages of a viral infection. ISG15, an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role, as well as can impart a direct antiviral effect against a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates an enhanced type-I IFN-mediated signaling, resulting in an increase in type-I IFN mediated antiviral effect during HEV replication. This observed enhanced type-I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in ISG15 mediated type-I IFN sensitivity against HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role, and regulates HEV-sensitivity to exogenous type-I IFN.

    IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, recently chronic HEV infection has become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid organ transplant patients with some successes. However, the mechanism behind type-I IFN mediated antiviral effect against HEV remains unclear. This study demonstrates ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type-I IFN signaling, and thus HEV-sensitivity to type-I IFN. Our results also show that PKR and OAS1 play an important role in ISG15 mediated type-I IFN sensitivity against HEV.

  • Translesion Polymerase, pol {eta}, is Required for Efficient Epstein-Barr Virus Infectivity and is Regulated by the Viral Deubiquitinating Enzyme, BPLF1. [PublishAheadOfPrint]

  • Epstein-Barr Virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. Previously we showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis pathway (TLS), a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument indicating that BPLF1 may function both early and late during infection. BPLF1 expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active-site cysteine results in loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (pol eta), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with pol eta, and that its protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of pol eta which are foci-like in appearance, consistent with the localization observed when pol eta is recruited to sites of DNA damage. Knockdown of pol eta resulted in decreased production of infectious virus, and further, pol eta was found to bind to EBV DNA suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as pol eta, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication.

    Importance: Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin lymphoma, Burkitt lymphoma and diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes, which is well studied but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein, and its conserved deubiquitinating activity, regulate the cellular DNA repair polymerase eta and recruits it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle; contributing to efficient infectious virion production.

  • Integrity of the linker of nucleoskeleton and cytoskeleton is required for efficient herpesvirus nuclear egress [PublishAheadOfPrint]

  • Herpesvirus capsids assemble in the nucleus while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain, and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection we generated cell lines constitutively expressing dominant-negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM.

    IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant-negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress, and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.

  • B cell-specific ATM expression promotes chronic gammaherpesvirus infection. [PublishAheadOfPrint]

  • Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in the host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloid-specific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM deficient B cells. Further, we found that during long term infection, peritoneal B-1b, but not related B-1a B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.

    SIGNIFICANCE Gammaherpesviruses infect a majority of human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of how these roles interplay in vivo, during chronic infection. In this study we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cell-specific ATM expression during chronic gammaherpesvirus infection.

  • Structure and functional basis for an EBNA1 hexameric ring in Epstein-Barr Virus episome maintenance [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the x-ray crystal structure of an EBNA1 DNA binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher order EBNA1 complexes. Substitution mutations around the interface destabilized higher order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. T585P was compromised for binding to OriP in vivo, as well as for assembling ORC2 and histone H3K4me3 at OriP. T585P was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymoprhisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1.

    IMPORTANCE Epstein-Barr virus is a human gamma herpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the life time of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows for long term persistence of the EBV genome are currently unclear. Here we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher order complex formation and long-term plasmid maintenance.

  • Experimental transmission of the chronic wasting disease agent to swine after oral or intracranial inoculation [PublishAheadOfPrint]

  • Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as a host for the agent of chronic wasting disease is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to one of three groups: intracranially inoculated (n=20), orally inoculated (n=19), or non-inoculated (n=9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled (llsquo;market weightrrsquo; groups). The remaining pigs (llsquo;agedrrsquo; groups) were allowed to incubate for up to 73 months post inoculation (MPI). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by western blotting (WB), antigen-capture immunoassay (EIA), immunohistochemistry (IHC) and in vitro real-time quaking induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC and/or WB. Using RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. Bioassay was positive in 4 out of 5 pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs using mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.

    IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months post-inoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results in orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.

  • Protection against HIV Acquisition in the RV144 Trial [PublishAheadOfPrint]

  • Differences of opinion regarding whether there may, or may not, have been protective efficacy in the RV144 vaccine trial have important societal implications.

  • Evaluation of the biological properties and cross-reactive antibody response to H10 influenza viruses in ferrets [PublishAheadOfPrint]

  • The recent outbreak of avian origin H10N7 influenza among seals in Northern Europe, and two fatal human infections with an avian H10N8 virus in China, have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound "avian-like" aalpha;2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain "human-like" aalpha;2,6-linked sialic acids, and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with gge; 14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against gge; 15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness.

    IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5-15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind "avian-like" sialic acids, although several isolates also displayed binding to "human-like" sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier, and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses, and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain.


  • Human papillomavirus (HPV) is a strongly conserved DNA virus, of which high-risk types can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A-D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole genome level. We have analyzed HPV16 major variant diversity, conservation in persistent infections and performed a SNP comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to three years. Our analysis shows a remarkably large variant diversity in the population. Whole genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified, where the initial and follow-up sample clustered differently. Non-A1/A2 variants seemed to clear preferentially (p=0.02). Our analysis shows population-wide HPV16 sequence diversity is very large. In persisting infections the HPV16 sequence is fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strong acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to three years of follow-up. These findings suggest progression of an early HPV16 infection could be host-related.

    IMPORTANCE Human papillomavirus (HPV) type 16 is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge on variants circulating in European populations is limited, especially with longitudinal follow-up. By sequencing a number of infections with known follow-up for up to three years, we gain initial insights in the genetic diversity of HPV16 and effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persisting infections did not identify strong-acting DNA variations responsible for these infection outcomes. In addition, we have identified a HPV16 reinfection event, where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency.

  • Increased, durable B-cell and ADCC Responses Associated with T-helper Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site. [PublishAheadOfPrint]

  • Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens for more long lived, efficacious HIV-1 vaccine induced B-cell responses. HIV-1 Env gp140 (native or un-cleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells may result in anergic effects on T-cell recruitment and consequently a lack of strong robust and durable B-memory responses. To test this hypothesis we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3kD CD4 miniprotein mimetic serving to block ligation of gp140 on CD4+T-cells while preserving CD4 inducible (CDi) neutralizing and epitopes targeted by antibody dependent cellular cytotoxic (ADCC) effector responses. Importantly immunization of rhesus macaques consistently gave superior B-cell (pllt;0.001) response kinetics and superior ADCC (pllt;0.014) in a group receiving the CD4bs-occluded vaccine compared to those animals immunized with gp140. Of the cytokines examined, Ag-specific IL-4 T-helper ELISpots in the CD4bs-occluded group increased earlier (p=0.025) during the inductive phase. Importantly CD4bs-occluded gp140 antigen not only induced superior B-cell and ADCC responses, the elevated B-cell responses proved to be remarkably durable lasting more than 60 weeks post-immunization.

    IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have until now been unsuccessful. Antigen specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B-cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T-cells as soluble shed antigen or as antigen antibody complexes causing impairment in the activation of these specialized CD4 positive T-cells. We proposed that CD4-binding impairment may in part be responsible for the relatively poor B-cell responses to HIV envelope based vaccines. To test this hypothesis we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

  • Selective expression of CCR10 and CXCR3 by circulating human HSV-specific CD8 T-cells [PublishAheadOfPrint]

  • Herpes simplex virus type (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T-cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia, and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T-cells. CLA/ESL mediates adhesion of T-cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChRs on HSV-specific human peripheral blood CD8 T-cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T-cells compared to CD8 T-cells specific for control viruses, EBV and CMV, and compared to bulk memory CD8 T-cells. CXCR3 ligand mRNA levels were selectively increased in recurrent HSV-2 skin biopsies, while the mRNA level of the CCR10 ligand CCL27 was equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T-cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.

    IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T-cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents and their expression of several candidate skin-homing associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are up-regulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.

  • Enterovirus 3A facilitates viral replication by promoting PI4KB-ACBD3 interaction [PublishAheadOfPrint]

  • Like other enteroviruses, EV71 relies on phosphatidylinositol-4-kinase IIIbbeta; (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor ACBD3 is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3 and 3A are all localized to the viral RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by siRNA leads to a reduction in the PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.

    IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme phosphatidylinositol-4-kinase IIIbbeta; by interacting with ACBD3, which alter cellular membranes through the production of a lipid PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide a new insight into the molecular network of enterovirus replication.

  • Partially uncleaved alphavirus replicase forms spherule structures in the presence and absence of RNA template [PublishAheadOfPrint]

  • Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein P1234, which is cleaved to four final products, nonstructural proteins nsP1-nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations, in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123, or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular compared to replication-induced spherules. In the presence of template, nsP1 + uncleaved P23 + nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membranes sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins.

    IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation and the different stages of RNA synthesis.

  • Propagation of astrovirus VA1, a neurotropic human astrovirus, in cell culture [PublishAheadOfPrint]

  • Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or in vivo models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that lead to discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 yielded increasing copies of VA1 RNA and multi-step growth curves demonstrated a greater than 100-fold increase in VA1 RNA 72 hours after inoculation. The full length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ~ 26 nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon response as VA1 RNA levels were reduced by pre-treatment of Caco-2 cells with interferon-bbeta;1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1.

    IMPORTANCE Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.

  • Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA prime - Modified Vaccinia Ankara (MVA) boost HIV-1 vaccine regimen [PublishAheadOfPrint]

  • Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function and specificity of Gag-specific T cells induced by a DNA-prime Modified Vaccinia Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding for a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55. Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced IFN-+ Gag-specific T cell responses were dominated by CD4+ T cells (compared to CD8+ T cells, pllt;0.001) that co-expressed IL-2 (66.4%) and/or TNFaalpha; (63.7%). A median of 3 antigenic regions were targeted with a higher median response magnitude to Gagp24 regions - more conserved between prime and boost - as compared to regions within Gagp15 (not primed) and Gagp17 (less conserved, both pllt;0.0001). Four regions within Gagp24 were each targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA and DNA Gag encoded immunogens (p=0.04, r2=0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 Protein.

    IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved; either by improved cross-recogniton of multiple variants for a given antigenic region or rather through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

  • HIV-1 Env- and Vpu-specific antibody-dependent cellular cytotoxicity responses associated with elite control of HIV [PublishAheadOfPrint]

  • Studying HIV-infected individuals who control HIV replication (elite controllers, ECs) enable exploration of effective anti-HIV immunity. HIV Env- and non-Env-specific antibody-dependent cellular cytotoxicity (ADCC) may contribute to protection from progressive HIV infection but the evidence is limited. We recruited 22 ECs and matched them with 44 viremic subjects. HIV Env- and Vpu-specific ADCC responses were studied in sera using a novel ELISA-based dimeric recombinant soluble (rs) FcRIIIa-binding assay, surface plasmon resonance, antibody-dependent natural killer (NK) cell activation assays and ADCC-mediated killing assays. ECs had higher levels of HIV Env-specific antibodies capable of binding FcRIIIa, activating NK cells and mediating Granzyme B activity (all pllt;0.01) compared to viremic subjects. ECs also had higher levels of antibodies against a C-terminal 13-mer Vpu peptide capable of mediating FcRIIIa-binding and NK cell-activation compared to viremic subjects (both pllt;0.05). Our data associate Env-specific and Vpu epitope-specific ADCC in effective immune responses against HIV among ECs. Our findings have implications for understanding the role of ADCC in HIV control.

    IMPORTANCE Understanding immune responses associated with elite control of HIV may aid the development of immunotherapeutic and vaccine strategies for controlling HIV infection. Env is a major HIV protein target of functional antibody responses that are heightened in ECs. Interestingly, EC antibodies also target Vpu, an accessory protein crucial to HIV, which degrades CD4 and antagonizes tetherin. Antibodies specific to Vpu are a common feature of the immune response of ECs that may prove of functional importance to the design of improved ADCC-based immunotherapy and preventative HIV vaccines.

  • Single-particle detection of transcription following rotavirus entry [PublishAheadOfPrint]

  • Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell-surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguish particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 minutes after adding virus. Uncoating efficiency was 20-50%; of the uncoated particles, about 10% synthesized detectable RNA. In the format of our experiments, about 1% of the added particles attached to the cell surface, giving an overall added-particle to RNA-synthesizing particle ratio of between 1000 and 5000 to 1, in good agreement with the particle-to-focus-forming unit determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.

    IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multi-step entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.

  • Effects of the NEDD8-activating enzyme inhibitor MLN4924 on lytic reactivation of Kaposi's sarcoma-associated herpesvirus [PublishAheadOfPrint]

  • The switch of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is a key event for viral dissemination and pathogenesis. MLN4924, a novel neddylation inhibitor, reportedly causes the onset of KSHV reactivation but impairs later phases of viral lytic program in infected cells. Thus far, the molecular mechanism involved in the modulation of KSHV lytic cycle by MLN4924 is not yet fully understood. Here, we confirmed that treatment of different KSHV-infected primary effusion lymphoma (PEL) cell lines with MLN4924 substantially induces viral lytic protein expression. Due to the key role of virally encoded ORF50 protein in the latent-to-lytic switch, we investigated its transcriptional regulation by MLN4924. We found that MLN4924 activates the ORF50 promoter (ORF50p) in KSHV-positive cells (but not in KSHV-negative cells), and the RBP-J-binding elements within the promoter are critically required for MLN4924 responsiveness. In KSHV-negative cells, reactivation of the ORF50 promoter by MLN4924 requires the presence of the latency-associated nuclear antigen (LANA). Under such a condition, LANA acts as a repressor to block the ORF50p activity, whereas MLN4924 treatment relieves the LANA-mediated repression. Importantly, we showed that LANA is a neddylated protein and can be deneddylated by MLN4924. On the other hand, we revealed that MLN4924 exhibits concentration-dependent biphasic effects on TPA- or sodium butyrate (SB)-induced viral reactivation in PEL cell lines. In other words, low concentrations of MLN4924 promote activation of TPA- or SB-mediated viral reactivation, whereas high concentrations of MLN4924 conversely inhibit the progression of TPA- or SB-mediated viral lytic program.

    IMPORTANCE MLN4924 is a neddylation (NEDD8 modification) inhibitor, which currently acts as an anti-cancer drug in clinical trials. Although MLN4924 has been reported to trigger KSHV reactivation, many aspects regarding the action of MLN4924 in regulating KSHV lytic cycle are not fully understood. Since KSHV ORF50 protein is the key regulator of viral lytic reactivation, we focus on its transcriptional regulation by MLN4924. We here show that activation of the ORF50 gene by MLN4924 involves the relief of LANA-mediated transcriptional repression. Importantly, we find that LANA is a neddylated protein. To our knowledge, this is the first report showing that neddylation occurs in viral proteins. Additionally, we provide evidence that different concentrations of MLN4924 have opposite effects on TPA-mediated or SB-mediated KSHV lytic-cycle activation. Therefore, in clinical application, we propose that MLN4924 needs to be used with caution in combination therapy to treat KSHV-positive subjects.

  • Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate ADCC, and Protect Mice from Ocular Challenge with HSV-1 [PublishAheadOfPrint]

  • The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus (HSV-1) glycoprotein D (gD), such that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV1 infection in cells expressing HVEM, but not the other major virus receptor- nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (a) neutralized HSV-1 infection in a cell receptor-specific manner, (b) mediated ADCC, and (c) reduced ocular disease in virus-infected mice.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores, neonatal herpes, and is a leading cause of blindness. Despite many trials, no HSV vaccine is approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were able to specifically neutralize HSV-1 infection in vitro via HVEM. Furthermore, we showed for the first time that HVEM-specific HSV-1 neutralizing antibodies protect mice from HSV-1 eye disease, indicating the critical role of HVEM in HSV-1 ocular infection.

  • Enterovirus 71 inhibits pyroptosis through cleavage of GSDMD [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) can cause hand, foot, and mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remain poorly understand. Here, we report that EV71 induces degradation of GSDMD, an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1-193. However, unlike the N-terminal fragment produced by casaspe-1 cleavage, this fragment fails to trigger cell death or inhibits EV71 replication. Importantly, T239D or F240D substitution abrogates the activity of GSDMD composed of amino acids 1-275. This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response.

    IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated IL-1bbeta; secretion. In this process, the N-terminal domain p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection down-regulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated Nmmdash;terminal fragment disrupted for inducing cell pyroptosis. Notably, the 1-275aa fragment (p30) inhibits EV71 replication whereas the 1-193aa fragment does not. These results reveal a new strategy for EV71 to evade the antiviral response.

  • Herpes Simplex Virus 1 Small Capsomere-Interacting Protein VP26 Regulates Nucleocapsid Maturation [PublishAheadOfPrint]

  • VP26 is a herpes simplex virus 1 (HSV-1) small capsomere-interacting protein. In this study, we investigated the function of VP26 in HSV-1-infected cells with the following results. (i) The VP26 null mutation significantly impaired incorporation of minor capsid protein UL25 into nucleocapsids (type C capsids) in the nucleus. (ii) The VP26 mutation caused improper localization of UL25 in discrete punctate domains containing multiple capsid proteins (e.g., the VP5 major capsid protein) in the nucleus: these domains corresponded to capsid aggregates. (iii) The VP26 mutation significantly impaired packaging of replicated viral DNA genomes into capsids, but had no effect on viral DNA concatemer cleavage. (iv) The VP26 mutation reduced the frequency of type C capsids, that contain viral DNA but not scaffolding proteins, and produced an accumulation of type A capsids, that lack both viral DNA and scaffold proteins, and had no effect on accumulation of type B capsids, that lack viral DNA but retain cleaved scaffold proteins. Collectively, these results indicated that VP26 was required for efficient viral DNA packaging, and proper localization of nuclear capsids. The phenotype of the VP26 null mutation was similar to that, reported previously, of the UL25 null mutation and of UL25 mutations that preclude its binding to capsids. Thus, VP26 appeared to regulate nucleocapsid maturation by promoting incorporation of UL25 into capsids, which is likely to be required for proper capsid nuclear localization.

    IMPORTANCE HSV-1 VP26 has been reported to be important for viral replication and virulence in cell cultures and/or mouse models. However, little is known about the function of VP26 during HSV-1 replication, in particular, in viral nucleocapsid maturation although HSV-1 nucleocapsids are estimated to contain 900 copies of VP26. In this study, we present data suggesting that VP26 promoted packaging of HSV-1 DNA genomes into capsids by regulating incorporation of capsid protein UL25 into capsids, which was reported to increase stability of the capsid structure. We also showed that VP26 was required for proper localization of capsids in the infected cell nucleus. This is the first report showing that HSV-1 VP26 is a regulator for nucleocapsid maturation.

  • SIVmac239, but not SIVmac316, binds and utilizes human CD4 more efficiently than rhesus CD4 [PublishAheadOfPrint]

  • Rhesus macaques are used to model HIV-1 infections, but they are not natural hosts of HIV-1 or any simian immunodeficiency virus (SIV). Rather, they became infected with SIV through cross-species transfer from sooty mangabeys in captivity. It has been shown that HIV-1 utilizes rhesus CD4 less efficiently than human CD4. However, the relative ability of SIV envelope glycoproteins to bind or utilize these CD4 orthologs has not been described. Here we show that several SIV isolates, including SIVmac239, are more efficiently neutralized by human CD4-Ig (huCD4-Ig) than by the same molecule bearing rhesus CD4 domains 1 and 2 (rhCD4-Ig). An I39N mutation in CD4 domain 1, present in human and sooty mangabey CD4 orthologs, largely restored rhCD4-Ig neutralization of SIVmac239 and other SIV isolates. We further observed that SIVmac316, a derivative of SIVmac239, bound to and was neutralized by huCD4-Ig and rhCD4-Ig with nearly identical efficiencies. Introduction of two SIVmac316 CD4 binding-site residues (G382R and H442Y) into the SIVmac239 envelope glycoprotein (Env) markedly increased its neutralization sensitivity to rhesus CD4-Ig without altering neutralization by human CD4-Ig, SIV neutralizing antibodies, or the sera of SIV-infected macaques. These changes also allowed SIVmac239 Env to bind rhCD4-Ig more efficiently than huCD4-Ig. The G382R/H442Y variant also infected cells expressing rhesus CD4 with markedly greater efficiency than did unaltered SIVmac239 Env. We propose that infections of rhesus macaques with SIVmac239 G382R/H442Y might better model some aspects of human infections.

    IMPORTANCE Rhesus macaque infection with simian immunodeficiency virus (SIV) has served as an important model of human HIV-1 infection. However, differences between this model and the human case have complicated the development of vaccines and therapies. Here we report the surprising observation that SIVmac239, a commonly used model virus, more efficiently utilizes human CD4 than the CD4 of rhesus macaques, whereas the closely related virus, SIVmac316 uses both CD4 orthologs equally well. We used this insight to generate a form of SIVmac239 envelope glycoprotein (Env) that utilized rhesus CD4 more efficiently, while retaining it resistance to antibodies and sera of infected macaques. This Env can be used to make the rhesus model more similar in some ways to human infection, for example by facilitating infection of cells with low levels of CD4. This property may be especially important to efforts to eradicate latently infected cells.

  • The host E3-Ubiquitin ligase TRIM6 ubiquitinates the Ebola virus VP35 protein and promotes virus replication [PublishAheadOfPrint]

  • Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central and West Africa. The EBOV genome encodes for VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type-I interferon (IFN-I) system. By using mass spectrometry analysis and co-immunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked poly-ubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated non-covalently with poly-ubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay, and TRIM6-knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.

    IMPORTANCE Ebola virus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40-90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a Tier 1 select agent risk group-4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination, and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.

  • A cell fusion-based screening method identifies the GPI-anchored protein Ly6e as the receptor for the mouse endogenous retroviral envelope syncytin-A [PublishAheadOfPrint]

  • Syncytins are envelope genes of retroviral origin that have been exapted for a role in placentation. They are involved in the formation of a syncytial structure (the syncytiotrophoblast) at the fetomaternal interface via their fusogenic activity. The mouse placenta is rather unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one, as observed in humans and all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryo survival. Their cognate cellular receptors, which are necessary to mediate cell-cell fusion and syncytiotrophoblast formation, are still unknown. By devising a sensitive method that combines a cell-cell fusion assay with the screening of a mouse cDNA library, we succeeded in identifying the GPI-anchored membrane protein Ly6e as a candidate receptor for SynA. Transfection of cells with the cloned receptor led to their fusion to cells expressing SynA, with no cross-reactive fusion activity with SynB. Knocking-down Ly6e greatly reduces SynA-induced cell fusion, thus suggesting that Ly6e is the sole receptor for SynA in vivo. Interaction of SynA with Ly6e was further demonstrated by a competition assay using the soluble ectodomain of Ly6e. Finally, RT-qPCR analysis of Ly6e expression on a representative panel of mouse tissues shows that it is significantly expressed in the mouse placenta together with SynA.

    IMPORTANCE Syncytins are envelope genes of endogenous retroviruses, coopted for a physiological function in placentation. They are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising a sensitive in vitro fusion assay that enables the high throughput screening of normalized cDNA libraries, we identified the long-sought receptor for syncytin-A (SynA), a mouse syncytin responsible for syncytiotrophoblast formation at the materno-fetal interface of the mouse placenta. This protein mmdash; Ly6e mmdash; is a GPI-anchored membrane protein, and siRNA experiments for its extinction as well as a decoy assay using a recombinant soluble receptor show that Ly6e is the necessary and sufficient partner of SynA. Its profile of expression is consistent with a role in both ancestral endogenization of a SynA founder retrovirus, and present-day placenta formation. This study provides a powerful general method to identify genes involved in cell-cell fusion processes.

  • Co-operation between strain-specific and broadly neutralizing responses limited viral escape, and prolonged exposure of the broadly neutralizing epitope. [PublishAheadOfPrint]

  • V3-glycan targeting broadly neutralizing antibodies (bNAbs) are a focus of HIV-1 vaccine development. Understanding the viral dynamics that stimulate the development of these antibodies can provide insights for immunogen design. We used a deep sequencing approach, together with neutralization phenotyping, to investigate the rate and complexity of escape from V3-glycan directed bNAbs, compared to overlapping early strain-specific neutralizing antibody (ssNAb) responses to the V3/C3 region in donor CAP177. Escape from the ssNAb response occurred rapidly, via a N334 to N332 glycan switch, which took just 7.5 weeks to reach ggt;50 % frequency. In contrast, escape from the bNAbs was mediated via multiple pathways and took longer, with escape first occurring through an increase in V1 loop length, which took 46 weeks to reach 50% frequency; followed by an N332 to N334 reversion which took 66 weeks. Importantly, bNAb escape was incomplete, with contemporaneous neutralization observed up to three years post infection. Both the ssNAb response and the bNAb response were modulated by the presence/absence of the N332 glycan, indicating an overlap between the two epitopes. Thus, selective pressure by ssNAbs to maintain the N332 glycan may have constrained the bNAb escape pathway. This slower and incomplete viral escape resulted in prolonged exposure of the bNAb epitope, which may in turn have aided the maturation of the bNAb lineage.

    IMPORTANCE: The development of an HIV-1 vaccine is of paramount importance, and broadly neutralizing antibodies are likely to be a key component of a protective vaccine. The V3-glycan targeting bNAb responses are among the most promising vaccine targets as they are commonly elicited during infection. Understanding the interplay between viral evolution and the development of these antibodies provides insights that may guide immunogen design. Our work contrasted the dynamics of the early strain-specific antibodies and the later broadly neutralizing responses, to a common Env target (V3C3), showing slower and more complex escape from bNAbs. Constrained bNAb escape, together with evidence of contemporaneous autologous virus neutralization, supports the proposal that prolonged exposure of the bNAb epitope enabled the maturation of the bNAb lineage.

  • Sec24C-Dependent Transport of Claudin-1 Regulates Hepatitis C Virus Entry [PublishAheadOfPrint]

  • Claudin-1 is an HCV co-receptor required for viral entry. Although extensive studies have focused on claudin-1 as an anti-HCV target, little is known about how the level of claudin-1 in cell surface is regulated by host vesicular transport. Here, we identified an interaction between claudin-1 and Sec24C, a cargo-sorting component of the COPII vesicular transport system. By interacting with Sec24C through its C-terminal YV, claudin-1 is transported from the ER and is eventually targeted into cell surface. Blocking COPII transport inhibits HCV entry by reducing the level of claudin-1 in cell surface. These findings provide mechanistic insight into the role of COPII vesicular transport in HCV entry.

    IMPORTANCE Tight junction protein claudin-1 is one of the cellular receptors for hepatitis C virus, which infected 185 million people globally. Its cellular distribution plays important role in HCV entry; however it is unclear how the localization of claudin-1 to cell surface is controlled by host transport pathways. In this paper, we not only identified Sec24C as a key host factor for HCV entry but also uncovered a novel mechanism by which the COPII machinery transports claudin-1 to cell surface. This mechanism might be extended to other claudins that contain C-terminal YV or V motifs.

  • Mutational analysis of Lassa virus glycoprotein highlights regions required for alpha-dystroglycan utilization [PublishAheadOfPrint]

  • Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable signal peptide, a receptor-binding subunit GP1, and a viral-host membrane fusion subunit GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (aalpha;DG) and lysosomal associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto VSV pseudo-particles and transduction efficiencies were monitored in HAP1 and HAP1-DAG1 cells that differentially produce the aalpha;DG cell surface receptor. Seven constructs retained efficient transduction in HAP1-DAG1 cells, yet poorly transduced HAP1 cells, suggesting they are involved in aalpha;DG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface, and are important for GP-aalpha;DG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-DAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is in common in both cell lines. Insight gained from these data can aid in the development of more effective entry inhibitors by blocking receptor interactions.

    Importance Countries with endemic Lassa virus such as Nigeria, Sierra Leone, Guinea and Liberia usually experience a seasonal outbreak of the virus from December to March. Currently, there is neither a preventative vaccine nor therapeutic available to effectively treat severe Lassa fever. One way to thwart virus infection is to inhibit interaction with cellular receptors. It is known that the GP1 subunit of the Lassa glycoprotein complex plays a critical role in receptor recognition. Our results highlight a region within the Lassa virus GP1 protein that interacts with the cellular receptor alpha-dystroglycan. This information may be used for future development of new Lassa virus antivirals.

  • Pleiotropic effects of resistance-breaking mutations on particle stability provide insight on life history evolution in a plant RNA virus [PublishAheadOfPrint]

  • In gene-for-gene host-virus interactions, virus evolution to infect and multiply in previously resistant host genotypes, i.e., resistance-breaking, is a case of host range expansion, predicted to be associated with fitness penalties. Negative effects of resistance-breaking mutations on within-host virus multiplication have been documented for several plant viruses. However, understanding virus evolution requires analyses of potential trade-offs between different fitness components. Here we analyze if coat protein (CP) mutations in Pepper mild mottle virus breaking L-gene resistance in pepper affect particle stability and, thus, survival in the environment. For this purpose, CP mutations determining the overcoming of L3 and L4 resistance alleles were introduced in biologically active cDNA clones. The kinetics of the in vitro disassembly of parental and mutants' particles was compared under different conditions. Resistance-breaking mutations variously affected particle stability. Structural analyses identified the number and type of axial and side interactions of adjacent CP subunits in virions, which explained differences in particle stability and contribute to understand tobamovirus disassembly. Resistance-breaking mutations also affected virus multiplication and virulence in the susceptible host, as well as infectivity. The sense and magnitude of the effects of resistance-breaking mutations on particle stability, multiplication, virulence or infectivity depended on the specific mutation, rather than on the ability to overcome the different resistance alleles, and effects on different traits were not correlated. Thus, results do not provide evidence of links or trade-offs between particle stability, i.e., survival, and other components of virus fitness, or virulence.

    IMPORTANCE The effect of survival on virus evolution remains underexplored, despite that life history trade-offs may constrain virus evolution. We approach this topic by analyzing if breaking of L-gene resistance in pepper by Pepper mild mottle virus, determined by coat protein (CP) mutations, is associated with reduced particle stability and survival. Resistance-breaking mutations affected particle stability by altering the interactions between CP subunits. However, the sense and magnitude of these effects were unrelated to the capacity to overcome different resistance alleles. Thus, resistance-breaking was not traded with survival. Resistance-breaking mutations also affected virus fitness within the infected host, virulence and infectivity in a mutation-specific manner. Comparison of the effects of CP mutations on these various traits indicates that there are neither trade-offs nor positive links between survival and other life history traits. These results demonstrate that trade-offs between life-history traits may not be a general constraint in virus evolution.

  • AAV serotypes have distinctive interactions with domains of the cellular receptor AAVR [PublishAheadOfPrint]

  • Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and proteinaceous receptor(s). Adeno-associated virus receptor (AAVR; also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes including the evolutionary distant serotypes, AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. Using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a 150-kDa molecular mass. By establishing a purification procedure, performing further protein separation through two-dimensional electrophoresis and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is N-linked glycosylated, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and viral overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like PKD repeat domain (PKD2) present in the ectodomain of AAVR. By contrast, AAV5 interacts primarily through the first, most membrane distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes including AAV1 and 8 require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.

    IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. Yet, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor, AAVR, is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate-limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor, and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discreet interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

  • On the death rate of abortively infected cells: estimation from simian/human immunodeficiency virus infection [PublishAheadOfPrint]

  • Progressive T cell depletion during chronic human immunodeficiency virus (HIV) infection is a key mechanism that leads to the development of AIDS. Recent studies have suggested that most T cells in the tissue die through pyroptosis triggered by abortive infection, i.e. infection of resting T cells where HIV failed to complete reverse transcription. However, the contribution of abortive infection to T cell loss and how quickly abortively infected cells die in vivo, key parameters for a quantitative understanding of T cell population dynamics, are not clear. Here, we infected rhesus macaques with simian-human immunodeficiency viruses (SHIV) and followed the dynamics of both plasma SHIV RNA and total cell-associated SHIV DNA. Fitting mathematical models to the data, we estimate that upon infection a majority of CD4+ T cells become abortively infected (approximately 65% on average) and die at a relatively fast rate of 0.27 day-1 (half-life: 2.6 days). This confirms the importance of abortive infection in driving T cell depletion. Further, we find evidence suggesting that an immune response maybe restricting viral infection 1-3 weeks after infection. Our study serves as a step-forward towards a quantitative understanding of the mechanisms driving T cell depletion during HIV infection.

    IMPORTANCE In HIV infected patients, progressive CD4+ T cell loss ultimately leads to the development of AIDS. The mechanisms underlying this T cell loss are not clear. Recent experimental data suggests that the majority of CD4+ T cells in tissue die through abortive infection where accumulation of incomplete HIV transcripts triggers cell death. To investigate the role of abortive infection in driving CD4+ T cell loss in vivo, we infected macaques with a simian-human immunodeficiency viruses (SHIV) and followed the viral kinetics of both plasma RNA and cell-associated DNA during infection. Fitting mathematical models, we estimated that a large fraction of infected cells die through abortive infection and have a half-life of approximately 2.6 days. Our results provide the first in vivo quantitative estimates of parameters characterizing abortive infection and supports the notion that abortive infection represents an important mechanism underlying progressive CD4+ T cell depletion in vivo.

  • Exploring and Exploiting Proteome Organization during Viral Infection [PublishAheadOfPrint]

  • Viral replication in eukaryotes is a process inherently organized in both space and time. Viral components target subcellular organelles to access host machineries required for replication and spread. Diverse viruses are known to alter organelle shape, composition, function, and dynamics as part of their replication cycles. Here, we highlight recent advances in microscopy and proteomic methods that have and will continue to help define mechanisms used by viruses to exploit host proteome organization.

  • West Nile virus NS1 antagonizes interferon-{beta} production by targeting RIG-I and MDA5 [PublishAheadOfPrint]

  • West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed significant a public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional non-structural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-bbeta; production, most likely through suppression of retinoic acid-inducible gene-I (RIG-I) like receptors (RLRs) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-bbeta; promoter after Sendai virus infection or polyinosinic-polycytidylic acid (poly(I:C)) treatment. NS1 also suppressed the activation of the IFN-bbeta; promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response.

    IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon-bbeta; (IFN-bbeta;) by interacting with and degrading retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation . This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.

  • Mumps Virus SH protein inhibits NF-{kappa}B activation by interacting with TNFR1, IL-1R1, and TLR3 complexes [PublishAheadOfPrint]

  • The mumps virus (MuV) small hydrophobic protein (SH) is a type I membrane protein expressed in infected cells. SH has been reported to interfere with innate immunity by inhibiting TNFaalpha;-mediated apoptosis and NF-B activation. To elucidate the underlying mechanism, we generated recombinant MuVs (rMuVs) expressing either the SH protein with an N-terminal FLAG epitope or lacking SH expression due to the insertion of three stop codons into the SH gene. Using these viruses, we were able to show that SH reduces the phosphorylation of IKKbbeta;, IBaalpha;, and p65 as well as the translocation of p65 into the nucleus of infected A549 cells. Reporter gene assays revealed that SH not only interferes with TNFaalpha;- but also with IL-1bbeta;- and poly(I:C)-mediated NF-B activation and that this inhibition occurs upstream of the NF-B pathway components TRAF2, TRAF6, and TAK1. Since SH co-immunoprecipitated with TNFR1, RIP1, and IRAK1, we hypothesize that SH exerts its inhibitory function by interacting with TNFR1, IL-1R1, and TLR3 complexes in the plasma membrane of infected cells.

    IMPORTANCE The MuV SH has been shown to impede with TNFaalpha;-mediated NF-B activation and is therefore thought to contribute to viral immune evasion. However, the mechanisms by which SH mediates NF-B inhibition remained largely unknown. In this study, we show that SH interacts with TNFR1, IL-1R1, and TLR3 complexes in infected cells. Thereby, we not only shed light onto the mechanisms of SH-mediated NF-B inhibition, but also reveal that SH interferes with NF-B activation induced by IL-1bbeta; and dsRNA.

  • Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus [PublishAheadOfPrint]

  • Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identified a 28-residue peptide (NP binding peptide, NPBP) from the Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18-344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP-core/NPBP complex at a resolution of 2.6 AAring; revealed that NPBP binds to the C-terminal region of the NP-core via electrostatic and non-polar interactions. Further structural analysis revealed that the MARV and EBOV NP-core holds a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP-core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from distinct termini between the MARV and EBOV NPBPs.

    IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP-core for NPBP across genera, a conserved pocket in the C-terminal region of the NP-core makes cross-species binding possible. Our results expand our knowledge of filovirus NP/VP35 interactions and provide more details for therapeutic intervention.

  • The nucleocapsid protein and non-structural protein 10 of highly pathogenic porcine reproductive and respiratory syndrome virus enhance CD83 production via NF-{kappa}B and Sp1 signaling pathways [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome, caused by the virus of the same name (PRRSV) is a panzootic disease, and one of the most economically costly to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs), and that the nucleocapsid (N) protein and non-structural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-B and Sp1 signaling pathways. Amino acid substitution mutants R43A and K44A of N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A of nsp10 abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A and rG214-3A) and four revertants (rR43A(R), rK44A(R), rP192-5A(R) and rG214-3A(R)) were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R) and rG214-3A(R). These findings suggest that PRRSV N protein and nsp10 play important roles in modulating CD83 signaling, and shed light on the mechanism by which PRRSV modulates host immunity.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host's immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-B and Sp1 signaling pathways. The viral nucleocapsid protein, non-structural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of N protein, as well as P192-5 and G214-3 of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

  • Case control comparison of enteric viromes in captive rhesus macaques with acute or idiopathic chronic diarrhea. [PublishAheadOfPrint]

  • Diarrhea is the major cause of non-research associated morbidity and mortality affecting the supply of rhesus macaques and, potentially, their responses to experimental treatments. Idiopathic chronic diarrhea (ICD) in rhesus macaques also resembles ulcerative colitis, a form of human inflammatory bowel disease. To test for viral etiologies we characterized and compared the fecal virome from 32 healthy, 31 with acute diarrhea, and 29 with ICD animals. The overall fraction of eukaryotic viral reads was 0.063% for the healthy group, 0.131% for the acute and 0.297% for the chronic diarrhea group. Eukaryotic viruses belonging to 6 viral families, as well as numerous circular Rep-encoding single stranded DNA (CRESS DNA) viral genomes, were identified. The most commonly detected sequences were from picornaviruses making up 59-88% of all viral reads followed by 9-17% for CRESS DNA virus sequences. The remaining 5 virus families: Adenoviridae, Astroviridae, Anelloviridae, Picobirnaviridae, and Parvoviridae collectively made up 1-3% of viral reads, except for parvoviruses which made up 23% of the viral reads in the healthy group. Detected members of the Picornaviridae and Parvoviridae families were highly diverse consisting of multiple genera, species, and genotypes. Co-infections with up to viral families were detected. Complete and partial viral genomes were assembled and used to measure the number of matching short sequence reads in feces from 92 animals in the two clinical and the healthy control groups. Several enterovirus genotypes and CRESS DNA genomes were associated with ICD relative to healthy animals. Conversely, higher reads numbers from different parvoviruses were associated with healthy animals. Our study reveals a high level of enteric co-infections with diverse viruses in a captive rhesus macaque colony and identifies several viruses positively or negatively associated with ICD.

  • Hepatitis C virus NS5A targets the nucleosome assembly protein NAP1L1 to control the innate cellular response [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral non-structural protein NS5A that, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified the nucleosome-assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirm this interaction and map it to the C-terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, but not from genotype 1, targets NAP1L1 for proteosomal-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling and we demonstrate the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those we show that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKK-mediated phosphorylation leading to inefficient RIG-I and TLR3 responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A.

    IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. The hepatitis C virus is capable of establishing a life-long chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study we identify a novel cellular target of the viral non-structural protein NS5A and demonstrate its role in antiviral signaling. This factor, called nucleosome-assembly protein like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide both evidence for a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.

  • Crystal structure and thermostability characterization of EV-D68-3Dpol [PublishAheadOfPrint]

  • Enterovirus-D68 (EV-D68) is one of the many non-polio enteroviruses that cause mild-to-severe respiratory illness. The non-structural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68, which plays a critical role in the replication of the viral genome and represents a promising drug target. Here we report the first three-dimensional crystal structure of RdRP from EV-D68 in complex with the substrate GTP to 2.3-AAring; resolution. The RdRP structure is similar to those of other viral RdRPs, where the three domains termed the palm, fingers, and thumb form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1-Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol. Interestingly, using limited proteolysis assays, we found that different NTPs stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterization conducted in this study helps us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well.

    IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68, and carried out systematic biophysical and biochemical characterization on the overall and local structural stability of the WT enzyme and several variants, which yields a clear view on the structure-activity relaitionship of the EV-D68 RNA polymerase.

  • Inhibition of Poxvirus Gene Expression and Genome Replication by Bisbenzimide Derivatives [PublishAheadOfPrint]

  • Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or anti-viral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole) and its derivatives, as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating post-replicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of-their-class, which appear to directly target the viral genome without affecting cell viability.

    IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus lifecycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and blocking viral replication.

  • Enfuvirtide (T20)-based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implication for Viral Entry and Inhibition [PublishAheadOfPrint]

  • The peptide drug Enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy of HIV-1 infection, but it has a relatively low antiviral activity and easily induces drug-resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors have greatly improved antiviral potency and in vivo stability, such as LP-11 and LP-19 that mainly target the gp41 pocket site. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target-binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity to the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry and infection. Contrary to LP-11 and LP-19, which required a flexible linker between the peptide sequence and lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying their different binding modes with the extended N-terminal helices of gp41. Also interestingly, LP-40 showed more potent activity over LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 to inhibit pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide has revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor but also revealed new insights into the mechanisms of viral inhibition.

    IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high dosages and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report that design of LP-40, a T20-based lipopeptide inhibitor which has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) do verify the current fusion model in which an extended pre-hairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

  • The Pace of Co-receptor Tropism Switch in HIV-1 Infected Individuals after Recent Infection [PublishAheadOfPrint]

  • HIV-1 entry into target cells influences several aspects of HIV-1 pathogenesis including viral tropism, HIV-1 transmission and disease progression and response to entry inhibitors. The evolution from CCR5 to CXCR4-using strains in a given human host is still unpredictable. Here we analyzed timing and predictors for co-receptor evolution among recently HIV-1 infected individuals. Proviral DNA was longitudinally evaluated in 66 individuals using Geno2Pheno[coreceptor]. Demographics, viral load, CD4+ and CD8+ T cell counts, CCR532 polymorphisms, GBV-C and HLA profiles were also evaluated. Ultradeep sequencing was performed on initial samples of 11 selected individuals. Tropism switch from CCR5 to CXCR4-using strains were identified in 9/49 (18.4%) individuals. Only low baseline false positive rate (FPR) was found to be a significant tropism switch predictor. No minor CXCR4-using variants were identified in initial samples of 4 of 5 R5/Non-R5 switchers. Logistic regression analysis showed that patients with an FPR ggt; 40.6% at baseline presented a stable FPR over time whereas lower FPRs tend to progressively decay leading to emergence of CXCR4-using strains, with a mean evolution time of 27.29 months (range: 8.90-64.62). An FPR threshold above 40.6% determined by logistic regression analysis may make unnecessary further tropism determination for prediction of disease progression related to emergence of X4 strains or use of CCR5 antagonists. The detection of variants with intermediate FPRs and its progressive decay over time not only strengthens the power of Geno2Pheno in predicting HIV tropism but also indirectly confirms a continuous evolution from earlier R5 variants towards CXCR4-using strains.

    IMPORTANCE Introduction of CCR5 antagonists in the antiretroviral arsenal has sparked interest in co-receptors utilized by HIV-1. Despite concentrated efforts, viral and human hosts' features predicting tropism switch are still poorly understood. Limited longitudinal data are available to assess the influence these factors have on predicting tropism switch and disease progression. The present study describes longitudinal tropism evolution in a group of recently HIV infected individuals, to determine the prevalence and potential correlates of tropism switch. We demonstrated here that low baseline FPR determined by the Geno2pheno[coreceptor] algorithm can predict tropism evolution from CCR5 to CXCR4 co-receptor use.

  • Differential innate immune signaling in macrophages by wild type vaccinia mature virus and a mutant virus deleting A26 protein [PublishAheadOfPrint]

  • The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of A26 protein from mature virus generates a mutant WRA26 that enters HeLa cells through plasma membrane fusion. Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type WR and WRA26 and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies in which HeLa cells infected by WR or WRA26 both expressed abundant viral late proteins, we found that WR expressed much less viral late protein than WRA26 in BMDM. Microarray analysis of cellular transcripts in BMDM induced by virus infection revealed that WR preferentially activated interferon receptor (IFNAR)-dependent signaling, but WRA26 did not. Consistently, we detected a higher level of soluble IFNbbeta; secretion and phosphorylation of Stat1 protein in BMDM infected with WR compared to WRA26. When IFNAR-knockout BMDM were infected with WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WR and in turn restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRA26 than WR infection, whereas IFNAR-knockout mice were equally susceptible to WR and WRA26 infection, demonstrating that the ability of WRA26 to evade IFNAR signaling has an important influence on viral pathogenesis in vivo.

    IMPORTANCE Vaccinia viral A26 protein was previously shown to mediate virus attachment and to regulate viral endocytosis. Here, we showed that WR infection induced a robust innate immune responses that activates interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas WRA26 did not. We further demonstrated that differential activation of IFNAR-dependent cellular signaling between WR and WRA26 is not only important for differential host restriction in BMDM but is also important for viral virulence in vivo. Our study reveals a new property of the WRA26 in regulating host antiviral innate immunity in vitro and in vivo.

  • Complete Genome Sequencing of Influenza A Viruses within Swine Farrow-to-Wean Farms Reveals the Emergence, Persistence, and Subsidence of Diverse Viral Genotypes [PublishAheadOfPrint]

  • Influenza A viruses (IAVs) are endemic in swine and represent a public health risk. However, there is limited information on the genetic diversity of swine IAVs within farrow-to-wean farms, which is where most pigs are born. In this longitudinal study, we sampled 5 farrow-to-wean farms during a year and collected 4,190 individual nasal swabs from three distinct pig subpopulations. 207 (4.9%) samples tested PCR positive for IAV, and 124 IAVs were isolated. We sequenced the complete genome of 123 IAV isolates, and found 31 H1N1, 26 H1N2, 63 H3N2 and 3 mixed IAVs. Based on the IAV hemagglutinin seven different influenza A viral groups (VGs) were identified. Most of the remaining IAV gene segments allowed us to differentiate the same VGs although an additional viral group was identified for gene segment 3 (PA). Moreover, the co-detection of more than one IAV VG was documented at different levels (farm, subpopulation, and individual pigs) highlighting the environment for potential IAV reassortment. Additionally, three out of 5 farms contained IAV isolates (n=5) with gene segments from more than one VG, and 79% of all IAVs sequenced contained a signature mutation (S31N) in the matrix gene that has been associated with resistance to the antiviral amantadine. Within farms, some IAVs were only detected once while others were detected for 283 days. Our results illustrate the maintenance and subsidence of different IAVs within swine farrow-to-wean farms over time, demonstrating that pig subpopulation dynamics is important to better understand the diversity and epidemiology of swine IAVs.

    IMPORTANCE At the global scale swine are one of the main reservoir species for influenza A viruses (IAVs), and play a key role on the transmission of IAVs between species. Additionally, the 2009 IAV pandemics highlighted the role of pigs in the emergence of IAVs with pandemic potential. However, limited information is available regarding the diversity and distribution of swine IAVs in farrow-to-wean farms where novel IAVs can emerge. In this study we studied 5 swine farrow-to-wean farms during a year and characterized the genetic diversity of IAVs among three different pig subpopulations commonly housed in this type of farms. Using next generation sequencing technologies, we demonstrated the complex distribution and diversity of IAVs among the pig subpopulations studied. Our results demonstrated the dynamic evolution of IAVs within farrow-to-wean farms, which is crucial to improve health interventions to reduce the risk of transmission between pigs and from pigs to people.

  • Human Bocavirus 1 Is a Novel Helper for Adeno-Associated Virus Replication [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1) is an autonomous parvovirus that infects well-differentiated primary human airway epithelia (HAE) in vitro. In human embryonic kidney (HEK) 293 cells, transfection of a duplex HBoV1 genome initiates viral DNA replication and produces progeny virions that are infectious in HAE. HBoV1 takes advantage of signaling pathways in the DNA damage response for efficient genome amplification in both well-differentiated (non-dividing) HAE and dividing HEK293 cells. On the other hand, adeno-associated virus 2 (AAV2) is a helper-dependent dependoparvovirus, and productive AAV2 replication requires co-infection with a helper virus (e.g., adenovirus or herpesvirus) or treatment with genotoxic agents. Here, we report that HBoV1 is a novel helper virus for AAV2 replication. HBoV1 and AAV2 co-infection rescued AAV2 replication in HAE cells. The helper function of HBoV1 for AAV2 is not only limited to HAE cells but also includes HEK293 and HeLa cells. Importantly, the HBoV1's helper function for AAV2 neither relies on HBoV1 replication nor DNA damage response. Following transfection of HEK293 cells, the minimal requirements for replication of AAV2 duplex DNA genome and production of progeny virions included HBoV1 NP1 and NS4 proteins, and the newly identified viral long non-coding RNA (BocaSR). However, following infection of HEK293 and HeLa cells with AAV2 virions, HBoV1 NS2 (but not NS4), NP1, and BocaSR were required for AAV2 DNA replication and progeny virion formation. These new methods for packaging AAV2 genome may be useful for generating recombinant AAV packaging cell lines and directed-evolution of AAV capsids.

    IMPORTANCE We first report that an autonomous parvovirus HBoV1 helps replication of a dependoparvovirus AAV2 in differentiated human airway epithelia. We identified the minimal sets of HBoV1 genes required to facilitate replication of AAV2 duplex genome and for AAV2 infection. Notably, together with expression of NP1 and BocaSR genes, HBoV1 NS2 is required for AAV2 productive infection in HEK293 and HeLa cells; whereas, NS4 is sufficient for viral DNA replication of an AAV2 duplex genome. The identification of HBoV1 as a helper virus for AAV2 replication has implications for improvement of recombinant AAV production in HEK293 cells and cell types that do not express adenovirus E1 gene, as well as for rescue of wild-type AAV genomes from tissues during directed-evolution in the absence of wild type adenovirus. Further understanding the mechanism underlying HBoV1 helper-dependent AAV2 replication may also provide insights into their functions in HBoV1 replication.

  • Phylogeography of coxsackievirus A16 reveals global transmission pathways and recent emergence and spread of a recombinant genogroup [PublishAheadOfPrint]

  • Coxsackievirus A16 (CV-A16; Picornaviridae) is an enterovirus (EV) type associated with hand-foot-and-mouth disease (HFMD) in children. To investigate the spatial spread of CV-A16, we used viral sequence data sampled during a prospective sentinel surveillance of HFMD in France (2010--2014) and phylogenetic reconstruction. A dataset of 168 VP1 sequences was assembled with 416 publicly available sequences of various geographic origins. The CV-A16 sequences reported were assigned to two clades, genogroup B and a previously uncharacterized clade D. The time origins of clades B and D were assessed in 1978 (1973--1981) and 2004 (2001--2007), respectively. The shape of the global CV-A16 phylogeny indicated worldwide co-circulation of genetically distinct virus lineages over time and across geographic regions. Phylogenetic tree topologies and Bayes factor analysis indicated virus migration. Virus transportation events were assessed in clade B, within Europe and Asia, and between countries of the two geographic regions. The sustained transmission of clade D viruses over 4 years was analysed at the township level in France and traced back to Peru, South America. Comparative genomics provided evidence of recombination between CV-A16 clades B and D, and suggested an intertype recombinant origin for clade D. Time-resolved phylogenies and HFMD surveillance data indicated that CV-A16 persistence is sustained by continuing virus migration at different geographic scales, from community transmission to virus transportation between distant countries. The results showed a significant impact of virus movements on the epidemiological dynamics of HFMD that could have implications for disease prevention.

    IMPORTANCE Coxsackievirus A16 is one of the most prevalent enterovirus types in hand-foot-and-mouth disease outbreaks reported in Southeast Asia. This study is based on epidemiological and viral data on HFMD caused by CV-A16 in a European country. The phylogeographic data complemented the syndromic surveillance with virus migration patterns between geographic regions in France. The results show how viral evolutionary dynamics and global virus spread interact to shape the worldwide pattern of an EV disease. CV-A16 transmission is driven by movements of infected individuals at different geographic levels: within a country (local dynamics), between neighboring countries (regional dynamics) and between distant countries (transcontinental dynamics). The results are consistent with our earlier data on EV-A71 and confirm the epidemiological interconnection of Asia and Europe regarding EV infections.

  • Transcriptional Profiling Confirms the Therapeutic Effects of Mast Cell Stabilization in a Dengue Disease Model [PublishAheadOfPrint]

  • There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage and shock due to the ability of DENV to infect a variety of immune and non-immune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs) and infected monocytes can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT and CD8+ T cell activation. The MC-stabilizing drug, ketotifen, reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immune-competent host and supports the further development of targeted immune-modulatory strategies to treat DENV disease.

    Importance: Dengue virus (DENV), which causes a febrile illness, is transmitted by mosquito vectors throughout tropical and sub-tropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection but it is thought that drugs that target the host's immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host's transcriptional response to infection in multiple DENV target organs using a mouse model of disease. We found that DENV infection induced metabolic dysregulation and inflammatory responses and affected the immune cell content of the spleen and liver. Use of the mast cell stabilization drug, ketotifen, reversed many of these responses, and induced additional changes in the transcriptome and immune cell repertoire that contribute to decreased dengue disease.

  • Cytomegalovirus Late Protein pUL31 Alters pre-rRNA Expression and Nuclear Organization during Infection [PublishAheadOfPrint]

  • The replication cycle of human cytomegalovirus (CMV) leads to drastic reorganization of domains in the host cell nucleus. However, the mechanisms involved and how these domains contribute to infection are not well understood. Our recent studies defining the CMV-induced nuclear proteome identified several viral proteins of unknown functions including a protein encoded by the UL31 gene. We set out to define the role of UL31 in CMV replication. UL31 is predicted to encode a 74 kDa protein, referred to as pUL31, containing a bi-partite nuclear localization signal, an intrinsically disordered region overlapping with arginine-rich motifs, and a C-terminal dUTPase-like structure. We observed that pUL31 is expressed with true-late kinetics and is localized to nucleolin-containing nuclear domains. However, pUL31 is excluded from the viral nuclear replication center. Nucleolin is a marker of nucleoli which are membrane-less regions involved in regulating ribosome biosynthesis and cellular stress responses. Other CMV proteins associate with nucleoli and we demonstrate that pUL31 specifically interacts with the viral protein, pUL76. Co-expression of both proteins altered pUL31 localization and nucleolar organization. During infection, pUL31 colocalizes with nucleolin but not the transcriptional activator, UBF. In the absence of pUL31, CMV fails to reorganize nucleolin and UBF and exhibits a replication defect at a low multiplicity. Finally, we observed that pUL31 is necessary and sufficient to reduce pre-rRNA levels and this was dependent on the dUTPase-like motif in pUL31. Our studies demonstrate that CMV pUL31 functions in regulating nucleolar biology and contributes to the reorganization of nucleoli during infection.

    IMPORTANCE Nucleolar biology is important during CMV infection with the nucleolar protein, nucleolin playing a role in maintaining the architecture of the viral nuclear replication center. However, the extent of CMV-mediated regulation of nucleolar biology is not well established. Proteins within nucleoli regulate ribosome biosynthesis and p53-dependent cellular stress responses that are capable of inducing cell cycle arrest and/or apoptosis, and are proposed targets for cancer therapies. This study establishes that CMV protein pUL31 is necessary and sufficient to regulate nucleolar biology involving the reorganization of nucleolar proteins. Understanding these processes will help define approaches to stimulate cellular intrinsic stress responses that are capable of inhibiting CMV infection.

  • A small viral PPxY-peptide motif to control antiviral autophagy [PublishAheadOfPrint]

  • Autophagy is an essential metabolic program also used for clearing intracellular pathogens. This mechanism, also termed selective autophagy, is well characterised for invasive bacteria but remains poorly documented for viral infections. Here we highlight our recent work showing that endosomolytic adenoviruses trigger autophagy when entering cells. Our study revealed how adenoviruses exploit a capsid-associated small PPxY peptide motif to manipulate the autophagic machinery to prevent autophagic degradation and to promote endosomal escape and nuclear trafficking.

  • Mitochondrial respiration is impaired during late stage hamster prion infection [PublishAheadOfPrint]

  • Mitochondria are crucial to proper neuronal function and overall brain health. Mitochondrial dysfunction within the brain has been observed in many neurodegenerative diseases including prion disease. Several markers of decreased mitochondrial activity have been reported during prion infection yet the bioenergetic respiratory status of mitochondria from prion-infected animals is unknown. Here, we show that clinically ill transgenic mice overexpressing hamster prion protein (Tg7) infected with the hamster prion strain 263K suffer from a severe deficit in mitochondrial oxygen consumption in response to the respiratory Complex II substrate, succinate. Characterization of the mitochondrial proteome of purified brain mitochondria from infected and uninfected Tg7 mice showed significant differences in the relative abundance of key mitochondrial electron transport proteins in 263K-infected animals relative to controls. Our results suggest that, at clinical stages of prion infection, dysregulation of respiratory chain proteins may lead to impairment of mitochondrial respiration in the brain.

    IMPORTANCE Mitochondrial dysfunction is present in most major neurodegenerative diseases and some studies have suggested that mitochondrial processes may be altered during prion disease. Here we show that hamster prion infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits. Tg7 mice infected with the 263K hamster prion strain have little or no signs of mitochondrial dysfunction at the disease midpoint but suffer from a severe deficit in mitochondrial respiration at the clinical phase of disease. A proteomic analysis of the isolated brain mitochondria from clinically affected animals showed that several proteins involved in electron transport, mitochondrial dynamics, and mitochondrial protein synthesis were dysregulated. These results suggest that mitochondrial dysfunction, possible exacerbated by prion protein overexpression, occurs at late stages during 263K prion disease and that this dysfunction may be the result of dysregulation of mitochondrial proteins.

  • Entry and release of hepatitis C virus in polarized human hepatocytes [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells. The relevance of cell polarity in the HCV life cycle has only been addressed in distant models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins, typical of columnar polarized epithelia which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on semi-permeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density was secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.

    IMPORTANCE Hepatitis C is a major health burden with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distant models and remains unclear. Hepatocyte organization is complex with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with a unique apical and basolateral domain was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower density viral particles were produced in conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.

  • Hepatitis C virus strain-dependent usage of apolipoprotein E modulates assembly efficiency and specific infectivity of secreted virions [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins and Apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE-dependency is common to all HCV genotypes remains unknown. Therefore, we compared the role of ApoE utilizing ten virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. RNAseq revealed abundant co-expression of ApoE, ApoB, ApoA1, A2, C1, C2, and C3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, E2, E3, A1, A2, C1 and C3) were provided in trans. All strains were strictly ApoE-dependent. However, ApoE involvement in virus production was strain- and cell type-specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and as ApoE knock-out differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, E3, E4) complemented virus production of HCV strains to a comparable degree. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, A2, C1, C3). The specific infectivity of HCV particles was similar between 293T and Huh-7.5-derived particles for most strains, however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry.

    IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable and strain-specific determinants modulate response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored if host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We show that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences not only relate to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage with implications for virus fitness at the level of assembly and cell entry.

  • Ifit2 is a Restriction Factor in Rabies Virus Pathogenicity [PublishAheadOfPrint]

  • Understanding the interactions between rabies virus (RABV) and individual host cell proteins is critical for the development of targeted therapies. Here we report that Interferon-induced protein with tetratricopeptide repeats 2 (Ifit2), an interferon-stimulated gene (ISG) with possible RNA-binding capacity, is an important restriction factor for rabies virus. When Ifit2 was depleted, RABV grew more quickly in mouse neuroblastoma in vitro. This effect was replicated in vivo, where Ifit2 knockout mice displayed a dramatically more severe disease phenotype than wild-type after intranasal inoculation of RABV. This increase in pathogenicity correlated to an increase in RABV mRNA and live viral load in the brain, as well as to an accelerated spread to brain regions normally affected by this RABV model. These results suggest that Ifit2 exerts its antiviral effect mainly at the level of viral replication, as opposed to functioning as a mechanism that restricts viral entry/egress or transports RABV particles through axons.

    IMPORTANCE Rabies is a fatal zoonotic disease with a nearly 100% case fatality rate. Although there are effective vaccines for rabies, this disease still takes the lives of about 50,000 people each year. Victims tend to be children living in regions without comprehensive medical infrastructure, presenting to health care workers too late for post-exposure prophylaxis. The protein discussed in our report, Ifit2, is found to be an important restriction factor for rabies virus, acting directly or indirectly against viral replication. A more nuanced understanding of this interaction may reveal a step of a pathway or site at which the system could be exploited for the development of a targeted therapy.

  • Analysis of HSV reactivation in explant reveals a method dependent difference in measured timing of reactivation [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) infection is widespread in the human population. Following orofacial infection, HSV establishes latency in innervating sensory neurons, primarily located in the trigeminal ganglia. A central feature of HSV pathogenesis is the ability to periodically reactivate in those neurons and transport back to the body surface. Both transmission and disease; such as keratitis, encephalitis, and neurodegeneration, have been linked to reactivation. Despite invaluable insights obtained from model systems, interactions between viral and host functions that regulate reactivation are still incompletely understood. Various assays are used for measuring reactivation in animal models, but there have been limited comparisons between methods and the accuracy of detecting the timing of reactivation and corresponding amount of infectious virus produced in the ganglia per reactivation event. Here, we directly compare two approaches for measuring reactivation in latently infected explanted ganglia by sampling media from the explanted cultures or homogenization of the ganglia and compare the results to viral protein expression in the whole ganglia. We show that infectious virus detection by direct homogenization of explanted ganglia correlates with viral protein expression, but detection of infectious virus in media samples from explanted cultures does not occur until extensive spread of virus is observed in the ganglia. The media sampling method is therefore not reflective of the initial timing of reactivation and the additional variables influencing spread of virus in the ganglia should be considered when interpreting results obtained using this method.

    IMPORTANCE The development of treatments to prevent and/or treat HSV infection rely upon understanding viral and host factors that influence reactivation. Progress is dependent on experimental methods that accurately measure the frequency and timing of reactivation in latently infected neurons. In this study, two methods for detecting reactivation using the explant model were compared. We show through direct tissue homogenization that reactivation occurs much earlier than can be detected by the indirect method of sampling media from explanted cultures. Thus, the sampling method does not detect the initial timing of reactivation and results obtained using this method are subject to additional variables with the potential to obscure reactivation outcomes.

  • Seasonal H3N2 and 2009 pandemic H1N1 influenza A viruses reassort efficiently but produce attenuated progeny [PublishAheadOfPrint]

  • Reassortment of gene segments between co-infecting influenza A viruses (IAV) facilitates viral diversification and has significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have co-circulated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous co-infections with pH1N1 and H3N2 viruses were compared to those obtained following co-infection with homologous, genetically tagged, pH1N1 viruses as a control. High genotype diversity was observed among progeny of both co-infections; however, diversity was more limited following heterologous co-infection. Pairwise analysis of genotype patterns revealed that homologous reassortment was random while heterologous reassortment was characterized by specific biases. pH1N1/H3N2 reassortant genotypes produced under single cycle co-infection conditions showed a strong preference for homologous PB2-PA combinations and general preferences for the H3N2 NA, pH1N1 M, and the H3N2 PB2 except when paired with the pH1N1 PA or NP. Multicycle co-infection results corroborated these findings and revealed an additional preference for the H3N2 HA. Segment compatibility was further investigated by measuring chimeric polymerase activity and growth of selected reassortants in human tracheobronchial epithelial cells. In guinea pigs inoculated with a mixture of viruses, parental H3N2 viruses dominated but reassortants also infected and transmitted to cage mates. Taken together, our results indicate that strong intrinsic barriers to reassortment between seasonal H3N2 and pH1N1 viruses are few, but that the reassortants formed are attenuated relative to parental strains.

    IMPORTANCE The genome of IAV is relatively simple, comprising eight RNA segments, each of which typically encodes one or two proteins. Each viral protein carries out multiple functions in coordination with other viral components and the machinery of the cell. When two IAV co-infect a cell, they can exchange genes through reassortment. The resultant progeny viruses often suffer fitness defects due to sub-optimal interactions among divergent viral components. The genetic diversity generated through reassortment can facilitate the emergence of novel outbreak strains. Thus, it is important to understand the efficiency of reassortment and the factors that limit its potential. The research described here offers new tools for studying reassortment between two strains of interest, and applies those tools to viruses of the 2009 pandemic H1N1 and seasonal H3N2 lineages, which currently co-circulate in humans and therefore have the potential to give rise to novel epidemic strains.

  • Discovery of a highly divergent coronavirus in the Asian house shrew from China illuminates the origin of the alphacoronaviruses [PublishAheadOfPrint]

  • Although shrews are one of the largest groups of mammals little is known about their role in the evolution and transmission of viral pathogens including coronaviruses. We captured 266 Asian house shrews (Suncus murinus) in Jiangxi and Zhejiang provinces, China, during 2013-2015. Coronavirus (CoV) RNA was detected in 24 Asian house shrews, with an overall prevalence of 9.02%. Complete viral genome sequences were successfully recovered from the RNA positive samples. The newly discovered shrew CoV fell into four lineages reflecting their geographic origins, indicative of largely allopatric evolution. Notably, these viruses were most closely related to alphacoronaviruses, but sufficiently divergent that they should be considered a novel member of the genus Alphacoronavirus, which we denote Weeacute;ncheeacute;ng shrew virus (WESV). Phylogenetic analysis revealed that WESV was a highly divergent member of the alphacoronaviruses and, more dramatically, that the S gene of WESV fell in a cluster that was genetically distinct from that of known coronaviruses. The divergent position of WESV suggests that coronaviruses have a long association with Asian house shrews. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to any known viruses. Together, these data suggest that shrews are natural reservoirs for coronaviruses and may have played an important and long-term role in CoV evolution.

    IMPORTANCE The subfamily Coronavirinae contains several notorious human and animal pathogens, including severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and porcine epidemic diarrhea virus. Because of their genetic diversity and phylogenetic relationships it has been proposed that the alphacoronaviruses likely have their ultimate ancestry in those viruses residing in bats. Here, we described a novel alphacoronavirus (Weeacute;ncheeacute;ng shrew virus, WESV) that was sampled from Asian house shrews in China. Notably, WESV is a highly divergent member of the alphacoronaviruses and possesses an S gene that is genetically distinct from that of all known coronaviruses. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to any known viruses. Together, these data suggest that shrews are important and long-standing hosts for coronaviruses that merit additional research and surveillance.

  • Functional evolution of influenza NS1 protein in currently circulating human 2009 pandemic H1N1 viruses [PublishAheadOfPrint]

  • In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist, but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here, we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of pH1N1 NS1 protein to naturally acquire mutations restoring this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and pro-inflammatory responses to a greater extent than the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. pH1N1/NSs-6mut virus grew similarly in mouse lungs to pH1N1/NSs-wt but infection with pH1N1/NSs-6mut induced lower levels of pro-inflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower inflammation induced by pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis.

    IMPORTANCE Seasonal influenza A viruses (IAV) are one of the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when (IAV) circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people until the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) allowing the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of pro-inflammatory cytokines, resulting in viral attenuation in vivo. This might represent an adaptation of pH1N1 virus to humans.

  • Interplay of PA-X and NS1 proteins in replication and pathogenesis of a temperature-sensitive 2009 pandemic H1N1 influenza A virus [PublishAheadOfPrint]

  • Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein has this inhibiting capability while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PAWT+/NS1MUT+) or do not have (PAMUT-/NS1WT-) the ability to block host gene expression showed reduced pathogenicity in vivo. However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PAMUT-/NS1MUT+) presented similar pathogenicity to a virus containing both wild-type proteins (PAWT+/NS1WT-). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV.

    IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition mechanisms, including prevention of host gene expression mediated by the viral PA-X and NS1 proteins. Here, we provide evidence demonstrating that optimal control of host protein synthesis by IAV PA-X and/or NS1 proteins is required for efficient IAV replication in the host. Moreover, we demonstrate the feasibility of genetically controlling the ability of IAV PA-X and NS1 proteins to inhibit host immune responses, providing an approach to develop more effective vaccines to combat disease caused by this important respiratory pathogen.

  • High Resolution Meta-Transcriptomics Reveals the Ecological Dynamics of Mosquito-Associated RNA Viruses in Western Australia [PublishAheadOfPrint]

  • Mosquitoes harbour a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a meta-transcriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes from the Aedes genus exhibiting substantially less diversity and lower abundance than those of Culex genus, within which viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95-98% nucleotide identity) to those from Indonesia and China. Finally, using abundance level and phylogenetic relationships we were able to distinguish potential mosquito viruses from those present in co-infecting bacteria, fungi, and protists. In sum, our meta-transcriptomics approach provides important insights into the ecology of mosquito RNA viruses.

    IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize meta-transcriptomic approaches to reveal both complete virus diversity and their relative abundance. We used such a meta-transcriptomic approach to determine key aspects of the ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are one of the most important components of the mosquito transcriptome, and we identified 19 new virus species from a diverse set of virus families. A key result was that host genetic background plays a more important role in shaping virus diversity than sampling location, with Culex species harbouring more viruses at greater abundance than those from Aedes mosquitoes.

  • Role of JNK pathway in varicella-zoster virus lytic infection and reactivation [PublishAheadOfPrint]

  • Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen activated protein kinases (MAPKs) have been shown to play a role in VZV infection of non-neuronal cells, with distinct consequences on infectivity in differing cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons, and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from acyclovir, and an acyclovir resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells, and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types, and may provide an alternative target for antiviral therapy.

    IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection, and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

  • Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection [PublishAheadOfPrint]

  • The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3rrsquo; exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3rrsquo; end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5rrsquo; end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved.

    IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved.

  • Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-driven Replication Capacity [PublishAheadOfPrint]

  • HIV infection is in the large majority of cases established by a single variant and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma derived Gag-protease from 53 non-transmitter mothers, 48 transmitter mothers and 47 infected infants were assayed in an HIV-1 inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacity between the non-transmitter (n=53) and transmitter mothers (n=44) (p = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than those derived from their mothers (p llt; 0.0001; paired T test). Higher percent similarity to consensus C Gag, p17, p24 and protease was also found in the infants (n = 28) in comparison to their mothers (p = 0.07, p = 0.002, p = 0.03 and p = 0.02 respectively; paired T test). These data suggest that of the viral quasispecies found in the mother, the HIV mother-to-child transmission bottleneck favours the transmission of consensus-like viruses with lower viral replication capacities.

    IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag - a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response - on the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability, as well as a higher similarity to the population consensus (in this case HIV subtype C), than that of their mothers. Furthermore, the observation that "consensus-like" virus sequences correspond to lower in vitro replication ability yet appear to be preferentially transmitted suggest that viral characteristics favouring transmission are decoupled from those that enhance replicative capacity.

  • Structural mimicry of the dengue virus envelope glycoprotein revealed by the crystallographic study of an idiotype-anti-idiotype Fab complex. [PublishAheadOfPrint]

  • A detailed understanding of the fine specificity of serotype-specific human antibodies is vital for the development and evaluation of new vaccines for pathogenic Flaviviruses such as Dengue virus (DENV) and Zika virus. In this study, we thoroughly characterize the structural footprint of an anti-idiotype antibody (E1) specific for a potent, fully human DENV serotype 1-specific antibody termed HM14c10, derived from a recovered patient. The crystal structure at a resolution of 2.5 AAring; of a complex between the Fab fragments of E1 and HM14c10 provides the first detailed molecular comparison of an anti-idiotype paratope specific for a human antibody with its analogous epitope- a discontinuous quaternary structure located at the surface of the viral particle that spans adjacent envelope (E) proteins. This comparison reveals that the footprints left by E1 and E on HM14c10 largely overlap, explaining why formation of the binary complexes are mutually exclusive. Structural mimicry of the DENV E epitope by the E1 combining site is achieved via the formation of numerous interactions with heavy chain CDRs of HM14c10, while fewer interactions are observed with its light chain, compared to the E protein. We show that E1 can be utilized to detect HM14c10-like antibodies in sera from patients recovered from a DENV-1 infection suggesting that this is a public (common) idiotype. These data demonstrate the utility of employing an anti-idiotype antibody to monitor a patient's specific immune responses and suggest routes for improvement of E llsquo;mimicryrrsquo; by E1 through increasing its recognition of the FabHM14c10 light chain CDRs.

    IMPORTANCE A chimeric yellow fever/dengue live-attenuated tetravalent vaccine is now marketed. Dengue remains a significant public health problem, because protection conferred by this vaccine is uneven against the four circulating serotypes. Reliable tools must be developed to measure the immune response of individuals exposed to DENV, either via viral infection or through vaccination. Anti-idiotypic antibodies provide precision tools for analyzing the pharmacokinetics of antibodies in an immune response and also for measuring the amount of circulating anti-infective therapeutic antibodies. Here, we characterize how an anti-idiotypic antibody (E1) binds the antibody HM14c10, which potently neutralizes DENV serotype 1. We report the crystal structure at a resolution of 2.5 AAring; of a complex between the Fab fragments of E1 and HM14c10 and provide the first detailed molecular comparison between the anti-idiotype surface and its analogous epitope located at the surface of the Dengue viral particle.

  • Genome-wide analysis of eighteen Epstein-Barr viruses isolated from primary nasopharyngeal carcinoma biopsies [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that is highly prevalent in almost all human populations, and is associated with many human cancers such as nasopharyngeal carcinoma (NPC), Hodgkin's disease, and gastric carcinoma. However, in these EBV-associated cancers, only NPC exhibits remarkable ethnic and geographic distribution. We hypothesized that EBV genomic variations might contribute to the pathogenesis of different human cancers in different geographic areas. In this study, we collected 18 NPC biopsies from the Hunan Province in Southern China and de novo assembled 18 NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated as HN1 to HN18. This was achieved through target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, to reveal sequence diversity. These EBV genomes harbored 20,570 variations totally, including 20,328 substitutions, 88 insertions, and 154 deletions, when compared to the EBV reference genome. Phylogenetic analysis revealed that all NPC-EBV genomes were distinct from other EBV genomes. Furthermore, HN1nndash;HN18 had some non-synonymous variations in EBV genes including those encoding latent, early lytic, and tegument proteins, such as substitutions within transmembrane domains 1 and 3 of LMP1, FoP_duplication, and zf-AD domains of ENBA1, in addition to aberrations in non-coding regions, especially in BamHI A rightward transcript microRNAs. These variations might have potential biological significance. In conclusion, we reported a genome-wide view of sequence variation in EBV isolated from primary NPC biopsy specimens obtained from the Hunan Province. This might contribute to further understanding of how genomic variations contribute to carcinogenesis, which would impact the treatment of EBV-associated cancer.

    IMPORTANCE Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV) infection and exhibits remarkable ethnic and geographic distribution. The Hunan Province of Southern China has a high incidence rate of nasopharyngeal carcinoma. Here, we report 18 novel Epstein-Barr virus genome sequences from viruses isolated from primary nasopharyngeal carcinoma biopsy specimens in this region, revealing whole-genome sequence diversity.

  • Differential requirement of human cytomegalovirus UL112-113 protein isoforms for viral replication [PublishAheadOfPrint]

  • The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear pre-replication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for recruitment of the viral UL84 and UL117 proteins. Remarkably, the p43 and p84 isoforms were both required for efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle.

    IMPORTANCE Human cytomegalovirus requires gene products from eleven genetic loci for lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N-termini by alternative splicing. In this study we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene product was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.

  • Going Against The Tide: Selective Cellular Protein Synthesis During Virally Induced Host Shutoff [PublishAheadOfPrint]

  • Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular energy generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology.

  • Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells [PublishAheadOfPrint]

  • Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles, in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are non-infectious and blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow for packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection.

    IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.

  • The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Expression of SUMO/sentrin Specific Peptidase 6 to Facilitate Latency Establishment [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV), belonging to -herpesviridae, typically displays two different phases in its lifecycle, the latent phase and the lytic phase. Latent-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of SUMO/sentrin specific peptidase 6 (SENP6) gene in vivo and in vitro. LANA could repress the SENP6 promoter activity at a dose dependent manner in the reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both RNA and protein levels. Moreover, SENP6 overexpression in KSHV infected cells reduced LANA at the protein level. Mechanically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which is relying on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would enhance viral gene expression with less abundance in LANA, which hampers the establishment of latency. Taken together, these data suggest that KSHV encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in the control of latency establishment.

    IMPORTANCE: LANA, as a key latent protein produced by KSHV, is responsible for the episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

  • Phosphorylation of serine 225 in hepatitis C virus NS5A regulates protein-protein interactions. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A, however the mechanistic details of the role of this post-translational modification in the virus life cycle remains obscure. We previously reported (Ross-Thriepland et al, 2015) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as Amphiphysin II) and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/western blotting, immunofluorescence and proximity ligation assay. Importantly, siRNA-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localisation of replication complexes.

    IMPORTANCE Hepatitis C virus is an important human pathogen. The viral non-structural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus lifecycle but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases and here we set out to determine whether this modification was required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they only interacted with NS5A that was phosphorylated on a specific residue. Furthermore these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

  • Human cytomegalovirus IE2 protein disturbs brain development by dysregulating neural stem cell maintenance and the polarization of migrating neurons [PublishAheadOfPrint]

  • Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days post-in utero-electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2+ cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed non-radially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2+ callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be respectively responsible for the increased neural stem cell exit from the VZ and cortical migrational defects. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.

    IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in the context of the embryonic brain, using an in utero gene transfer tool. Surprisingly, IE2, but not HCMV IE1 or MCMV ie3, interferes pleiotropically with key neurodevelopmental processes including neural stem cell regulation, proper positioning of migrating neurons, and the callosal axon projections important for communication between the hemispheres. Our data suggest that the wide spectrum of clinical outcomes ranging from mental retardation to microcephaly caused by congenital HCMV infection can be sufficiently explained in terms of IE2 action alone.

  • Conserved V{delta}1 binding geometry in a setting of locus-disparate pHLA recognition by {delta}/{alpha}{beta}TCRs: insight into recognition of HIV peptides by TCR [PublishAheadOfPrint]

  • Given a limited set of TCR V genes which are used to create TCRs that are reactive to different ligands, such as MHC class I, MHC class II and MHC-like proteins (for example, MIC molecules and CD1 molecules), the V1 segment can be rearranged with D-J-C or Jaalpha;-Caalpha; segments, to form classical TCR or uncommon aalpha;bbeta;TCR using a V1 segment (/aalpha;bbeta;TCR). Here we have determined two complex structures of the /aalpha;bbeta;TCRs (S19-2 and TU55) bound to different locus-disparate MHCIs with HIV peptides (HLA-A*2402-Nef138-10 and HLA-B*3501-Pol448-9). The overall binding modes resemble classical aalpha;bbeta;TCRs, but display a strong tilt binding geometry of V1 domain towards the HLA aalpha;1 helix, due to a conserved extensive interaction between the CDR1 loop and N-terminal region of aalpha;1 helix (mainly in position 62). The aromatic amino acids of the CDR1 loop exploit different conformations ("aromatic-ladder" or "aromatic-hairpin") to accommodate distinct MHC helical scaffolds. This tolerance helps to explain how a particular TCR V region can similarly dock onto multiple MHC molecules, and thus, may potentially explain the nature of TCR cross-reactivity. In addition, the length of CDR3 loop could affect the extent of tilt binding of V1 domain, and adaptively, the pairing Vbbeta; domains adjust their mass centers to generate differential MHC contacts, hence probably ensuring the TCR specificity to a certain peptide-MHC. Our data have provided further structural insights into the TCR recognition of classical pMHCI molecules, unifying the cross-reactivity and specificity together.

    IMPORTANCE The specificity of aalpha;bbeta; T cell recognition is determined by the CDR loops of the aalpha;bbeta;TCR and the general binding mode of aalpha;bbeta;TCRs to pMHC has been established over the last decade. Due to the intrinsic genomic structure of the TCR aalpha;/ chain locus, some V segments can rearrange with Caalpha; segment, forming a hybrid VCaalpha;Vbbeta;Cbbeta; TCR, /aalpha;bbeta;TCR. However, the basis for the molecular recognition of such TCRs to their ligands is elusive. Here, an aalpha;bbeta;TCR using V1 segment, S19-2, is isolated from a HIV-infected patient, in an HLA-A*24:02 restricted manner. Then we solved the crystal structures of S19-2 TCR and another /aalpha;bbeta;TCR TU55 binding to their ligands respectively, revealing a conserved V1 binding feature. Further binding kinetics analysis reveals that the S19-2 and TU55 TCRs bind pHLA very tightly and long-lastingly. Our results illustrate the binding mode of a TCR using V1 segment to its ligand, virus-derived pHLA.

  • Microtubule regulation and function during virus infection [PublishAheadOfPrint]

  • Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes including the capture, transport and spatial organization of cargos and organelles, as well as changes in cell shape, polarity and motility. Nucleating from MT organizing centers including, but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing post-translational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated Proteins (MAPs) that include a subset of highly specialized plus end-tracking proteins (+TIPs), which respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.

  • Vesicular stomatitis virus pseudotyped with Ebola virus glycoprotein serves as a protective, non-infectious vaccine against Ebola virus challenge in mice [PublishAheadOfPrint]

  • The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA-approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor binding domain (RBD) thereby blocking epitopes within the RBD, we also tested if VSVG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than WT GP/VSVG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, protection conferred correlated with the quantity of EBOV GP-specific Ig produced, but not the production of neutralizing antibodies. Our results show that EBOV GP/VSVG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and GP1 N-glycan loss does not influence immunogenicity or vaccination success.

    IMPORTANCE The West African Ebola virus epidemic was the largest to date with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVG pseudovirion vaccines may prove as efficacious and have better safety, but have not been tested to date. Thus, we test the efficacy of VSVG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV challenged mice. Further, we found removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

  • Intrasubtype B HIV-1 Superinfection Correlates with Delayed Neutralizing Antibody Response [PublishAheadOfPrint]

  • Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures, and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART) naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to six months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against Tier 1 subtype B viruses (p = 0.003 for SF-162 and p = 0.017 for NL4-3) and marginally against autologous virus (p = 0.054). Lower pre-superinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between two and three years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by pre-superinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary-infecting and Tier 1 subtype B viruses may be more susceptible to superinfection.

    IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some superinfected individuals. These findings will inform HIV vaccine design by providing testable correlates of protection from initial HIV infection.

  • Follicular regulatory T cells are highly permissive to R5-tropic HIV-1 [PublishAheadOfPrint]

  • Follicular regulatory T cells (TFR) are a subset of CD4+ T cells in secondary lymphoid follicles. TFR were previously included in the follicular helper T cell (TFH) subset, which are highly permissive to HIV-1. The permissivity of TFR to HIV-1 is unknown. We find TFR are more permissive than TFH to R5-tropic HIV-1 ex vivo. TFR expressed more CCR5 and CD4, and supported higher frequencies of viral fusion. Differences in Ki67 expression correlated with HIV-1 replication. Inhibiting cellular proliferation reduced Ki67 expression and HIV-1 replication. Lymph node cells from untreated HIV-infected individuals revealed that TFR harbored the highest concentrations of HIV-1 RNA and highest levels of Ki67 expression. These data demonstrate that TFR are highly permissive to R5-tropic HIV-1 both ex vivo and in vivo. This is likely related to elevated CCR5 levels combined with a heightened proliferative state and suggests TFR contribute to persistent R5-tropic HIV-1 replication in vivo.

    Importance In chronic, untreated HIV-1 infection, viral replication is concentrated in secondary lymphoid follicles. Within secondary lymphoid follicles, follicular helper T cells (TFH) have previously been shown to be highly permissive to HIV-1. Recently, another subset of T cells in secondary lymphoid follicles was described, follicular regulatory T cells (TFR). These cells share some phenotypic characteristics with TFH and studies that showed that TFH are highly permissive to HIV-1 included TFR in their definition of TFH. The permissivity of TFR to HIV-1 has not previously been described. Here, we show that TFR are highly permissive to HIV-1 both ex vivo and in vivo. The expression of Ki67, a marker of proliferative capacity, is predictive of expression of viral proteins and downregulating Ki67 leads to concurrent decreases in expression of viral proteins. Our study provides new insight into HIV-1 replication and a potential new cell type to target for future treatment.

  • Kallikrein-related peptidase 5 contributes to H3N2 influenza virus infection in human lungs. [PublishAheadOfPrint]

  • The HA (hemagglutinin) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies have indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating the HA of seasonal influenza A viruses (IAV) in humans. This study focuses on human KLK1 and KLK5, two of the 15 serine proteases known as the kallikrein-related peptidases (KLK). We find that their mRNA expression in primary human bronchial cells is stimulated by an IAV infection. Both enzymes cleaved recombinant HA from several strains of H1 and/or H3 virus subtypes in vitro, but only KLK5 promoted the infectivity of A/PR/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. Nasal instillation of only KLK5-treated virus resulted in weight loss and lethal outcome. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions by a KLK5-selective inhibitor significantly reduced activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans.

    IMPORTANCE Influenza A viruses (IAV) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered as promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient to promote infectivity of H3N2 IAV in vitro and in vivo. Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by a H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.

  • Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer [PublishAheadOfPrint]

  • We recently showed that mutations in the HIV-1 Envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (mAbs). Here we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wildtype JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 mAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intra-protomer interactions exposed epitopes in the V2i region (overlays the aalpha;4bbeta;7 integrin binding site) and the V3 crown, suggesting a general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutants affecting inter-protomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These latter data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed mAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together these data indicate that several transient Env epitopes can be rendered more accessible to Abs via specific mutations, and this may facilitate design of V1V2-targeting immunogens.

    Importance Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine trial correlated with the elicitation of V2- and V3-directed antibodies. Previously we identified various mechanisms responsible for destabilizing the V3 loop; here we determined, via mutation of numerous Env residues, which of these elements maintain the V1V2 loop in an inaccessible state and which expose V1V2 and/or V3 epitopes. Notably, our data indicate that V3 can move independently of V2, but none of the mutations studied expose V2 epitopes without also exposing V3. Additionally, V1V2 can be rendered more accessible to Abs via specific mutations, facilitating the development of engineered V2 immunogens.

  • In-vitro replication of Chelonid herpesvirus 5 in organotypic skin cultures from Hawaiian green turtles (Chelonia mydas) [PublishAheadOfPrint]

  • Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with Chelonid herpesvirus 5 (ChHV5) that has historically been refractory to growth in tissue culture. Here, we show for the first time de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative for active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included 1) either in-vitro culturing of ChHV5-positive tumor biopsies (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and 2) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies revealing intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegumentation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign for active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures where most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as model to culture other viruses that are resistant to replication in conventional cell culture.

    Importance: A major challenge in virology is viruses that cannot be grown in the laboratory. One example is Chelonid herpesvirus 5 (ChHV5) associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathology shows that ChHV5 is shed in skin. Here, we show that ChHV5 will grow in vitro if we replicate the complex three- dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, morphogenesis of herpesviral growth in three dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have application to other viruses including those of humans.

  • Deficiency of the NOD-like receptor NLRC5 results in decreased CD8+ T cell function and impaired viral clearance [PublishAheadOfPrint]

  • Pathogen recognition receptors are vital components of the immune system. Engagement of these receptors is important not only for instigation of innate immune responses to invading pathogens but also for initiating the adaptive immune response. Members of the NOD-like receptor (NLR) family of pathogen recognition receptors have important roles in orchestrating this response. The NLR family member, NLRC5 regulates major histocompatibility complex class I (MHC-I) expression during various types of infections, but its role in immunity to influenza A virus (IAV) is not well studied. Here, we show that Nlrc5-/- mice exhibit an altered CD8+ T cell response during IAV infection compared to wild type (WT) mice. Nlrc5-/- mice have decreased MHC-I expression on hematopoietic cells and fewer CD8+ T cells prior to infection. NLRC5 deficiency did not affect generation of antigen specific CD8+ T cells following IAV infection; however, a change in epitope dominance was observed in Nlrc5-/- mice. Moreover, IAV specific CD8+ T cells from Nlrc5-/- mice have impaired effector functions. This change in the adaptive immune responses is associated with impaired viral clearance in Nlrc5-/- mice. Collectively, our results demonstrate an important role for NLRC5 in regulation of antiviral immune responses and virus clearance during IAV infection.

    IMPORTANCE The NOD-like receptor family member NLRC5 is known to regulate expression of MHC-I as well as other genes required for antigen processing. In addition, NLRC5 also regulates various immune signalling pathways. In this study, we investigated the role of NLRC5 during influenza virus infection and found a major role for NLRC5 in restricting virus replication and promoting viral clearance. The observed increase in viral titers correlated with impaired effector CD8+ T cell responses. Although NLRC5-deficient mice were defective in clearing the virus, they did not show increase in morbidity or mortality following influenza virus infection because of other compensatory immune mechanisms. Therefore our study highlights how NLRC5 regulates multiple immune effector mechanisms to promote host defence during influenza virus infection.

  • Polymorphic Nature of HTLV-1 Particle Cores as Revealed Through Characterization of a Chronically Infected Cell Line [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. HTLV-1 particle structure is still poorly understood, and previous studies have analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically-infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of HTLV-1 particle structure. First, we identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. Furthermore, we determined that despite encoding for a truncated Gag, only full-length Gag protein is incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed 3 classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal in shape, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed from MT-2 cells, and provide evidence that the known poor infectivity of HTLV-1 particles correlates with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure.

    IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically-infected cell line are polymorphic in nature, with many particles lacking an organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.

  • A conserved leucine zipper motif in gammaherpesvirus ORF52 is critical for distinct microtubule rearrangements [PublishAheadOfPrint]

  • Productive viral infection often depends on manipulation of the cytoskeleton. Herpesviruses, including Rhesus monkey rhadinovirus (RRV) and its close homolog the oncogenic human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), exploit microtubule-based retrograde transport to deliver their genomes to the nucleus. Subsequently, during the lytic phase of the life cycle, the maturing viral particles undergo orchestrated translocation to specialized regions within the cytoplasm, leading to tegumentation, secondary envelopment, and then egress. As a result, we hypothesized that RRV might induce changes in the cytoskeleton at both early and late stages of infection. Using confocal imaging, we found that RRV infection led to thickening and acetylation of microtubules (MTs) emanating from the MT organizing center (MTOC) shortly after viral entry and more pronounced and diffuse MT reorganization during peak stages of lytic gene expression and virion production. We subsequently identified ORF52, a multifunctional and abundant tegument protein, as being the only virally encoded component responsible for these cytoskeletal changes. Mutational and modeling analyses indicated that an evolutionarily conserved, truncated leucine zipper motif near the N-terminus as well as a strictly conserved arginine toward the C-terminus of ORF52 play critical roles in its ability to rearrange the architecture of the MT cytoskeleton. Taken together, our findings combined with earlier studies describing diverse roles for ORF52 suggest that it likely binds to different cellular components, thereby allowing context-dependent modulation of function.

    IMPORTANCE A thorough understanding of the processes governing viral infection includes knowing how viruses manipulate their intracellular milieu, including the cytoskeleton. Altering dynamics of actin or MT polymerization, for example, are common strategies viruses employ to ensure efficient entry, maturation, and egress, as well as avoidance of antiviral defenses through the sequestration of key cellular factors. We found that infection with RRV, a homolog of the human pathogen KSHV, led to peri-nuclear wrapping by acetylated MT bundles and identified ORF52 as the viral protein underlying these changes. Remarkably, incoming virions were able to supply sufficient ORF52 to induce MT thickening and acetylation near the MTOC, potentially aiding in viral genome delivery to the nucleus. Although the function of MT alterations during the late stages of infection requires further study, ORF52 does share functional and structural similarities to alphaherpesvirus VP22, underscoring the evolutionary importance of MT cytoskeletal manipulations for this virus family.

  • Impaired downregulation of NKG2D ligands by Nef protein from elite controllers sensitizes HIV-1-infected cells to ADCC [PublishAheadOfPrint]

  • HIV-1 Nef clones isolated from a rare subset of HIV-1-infected elite controllers (EC), with the ability to suppress viral load to undetectable levels in the absence of antiretroviral therapy, are unable to fully downregulate CD4 from the plasma membrane of CD4+ T cells. Residual CD4 left at the plasma membrane allows Env-CD4 interaction, which leads to increased exposure of Env CD4-induced epitopes and increases susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated largely by natural killer (NK) cells, which control their activation status through the cumulative signals received through activating and inhibitory receptors. Recently, the activating NKG2D receptor was demonstrated to positively influence ADCC responses. Since HIV-1 Nef has been reported to reduce the expression of NKG2D ligands, we evaluated the relative abilities of Nef from EC and progressors to downmodulate NKG2D ligands. Furthermore, we assessed the impact of EC and progressor Nef on the ADCC susceptibility of HIV-1-infected cells. We observed a significantly increased expression of NKG2D ligands on cells infected with viruses coding for Nef from EC. Importantly, NKG2D ligand expression levels correlated with enhanced susceptibility of HIV-1-infected cells to ADCC. The biological significance of this correlation was corroborated by the demonstration that antibody-mediated blockade of NKG2D significantly reduced ADCC of cells infected with viruses carrying Nef from EC. These results suggest the involvement of NKG2D/NKG2D ligand interactions in the enhanced susceptibility of EC HIV-1-infected cells to ADCC responses.

    IMPORTANCE Attenuated Nef functions have been reported in HIV-1 isolated from elite controllers (EC). The inability of elite controller Nef to fully remove CD4 from the surface of infected cells enhanced their susceptibility to elimination by ADCC. We now show that downregulation of NKG2D ligands by HIV-1 Nef from EC is inefficient and leaves infected cells susceptible to ADCC. These data suggest a critical role for NKG2D ligands in anti-HIV-1 ADCC responses.

  • microRNAs of Epstein-Barr virus control innate and adaptive anti-viral immunity [PublishAheadOfPrint]

  • More than 90% of humanity is infected with Epstein-Barr virus (EBV) lifelong. While infection is usually controlled by the immune system, the human host fails to completely eliminate the pathogen. Several herpesviral proteins are known that act as immunoevasins preventing or reducing recognition of EBV-infected cells. Only recently, microRNAs of EBV were identified to reduce immune recognition further. This Gem summarizes what we know about immunomodulatory miRNAs of herpesviruses.

  • Role of autophagy in the HIV-1 matrix protein p17-driven lymphangiogenesis [PublishAheadOfPrint]

  • AIDS-related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV-1-infected (HIV+) patients, but does not affect the occurrence of ARLs to the same extent as of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV-1 does not infect the neoplastic B cells, but HIV-1 proteins have been hypothesized to play a key role in sustaining a pro-lymphangiogenic microenvironment in lymphoid organs. The HIV-1 matrix protein p17 (p17) is detected in blood and accumulates in the germinal centers of lymph nodes of HIV+ patients under successful cART. The viral protein displays potent lymphangiogenic activity in vitro and in vivo. This was, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin-1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here we show that the p17 lymphangiogenic activity occurs on human lymph node-derived lymphatic endothelial cells (LN-LECs) under stress conditions only and relies entirely on activation of an autophagy-based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibited p17-triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy-incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor-driven lymphangiogenesis in HIV+ patients.

    IMPORTANCE AIDS-related lymphomas (ARLs) are the most common malignancies in HIV-1-infected (HIV+) patients after the introduction of the combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV+ patients under successful cART. The HIV-1 matrix protein p17 (p17) is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several data suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here we show that p17 promotes lymphangiogenesis of human lymph node-derived lymphatic endothelial cells (LN-LECs). The lymphangiogenic activity of p17 is sustained by an autophagy-based pathway that enables LN-LECs to release pro-lymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.

  • HIV-1 Epitope Variability Is Associated With T Cell Receptor Repertoire Instability and Breadth [PublishAheadOfPrint]

  • Mutational escape of HIV-1 from HIV-1-specific CD8+ T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia llt;3000 RNA copies/ml over 17 to 179 weeks. The composition of TCRs targeting each epitope varied considerably in stability over time, although clonal stability (Sorensen index) was not significantly time-dependent within this interval. However, TCR stability inversely correlated to epitope variability in the Los Alamos HIV-1 Sequence Database, consistent with TCR evolution being driven by epitope variation. Finally, a robust inverse correlation of TCR breadth against each epitope versus epitope variability further suggested that this variability drives TCR repertoire diversification. In the context of studies demonstrating rapidly shifting HIV-1 sequences in vivo, our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution, and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.

    IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8+ T lymphocytes (CTLs) is a major barrier for effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here we demonstrate that the clonal stability and number of clones for the CTL response against an epitope is inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV-1 escape may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.

  • Newcastle disease virus establishes persistent infection in tumor cells in vitro: contribution of the cleavage site of fusion protein and second sialic acid binding site of hemagglutinin-neuraminidase [PublishAheadOfPrint]

  • Newcastle Disease Virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of an ovarian human cancer cell line (OVCAR3) with a recombinant low pathogenic NDV, persistent infection (PI) was established in a subset of tumor cells. PI cells exhibited resistance to superinfection with NDV, and an anti-viral state as demonstrated by upregulation of interferon and interferon induced genes such as Myxoma resistance gene 1 (Mx1) and Retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell to cell fusion following infection compared to the parental virus in two tumor cell lines tested, HT1080 and HeLa, and remained to be attenuated in chickens. Two mutations, one in fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R) located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency facilitating cell-to-cell fusion, while HN169R possesses a multi-faceted role in contributing to higher fusion, reduced receptor binding and lower neuraminidase activity, together resulting in increased fusion activity and reduced viral replication. Thus, establishment of PI in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host anti-viral responses. The results of our study also demonstrate a critical role of the second receptor binding region in the HN protein, which is conserved in several paramyxoviruses, in viral life cycle.

    IMPORTANCE Oncolytic Newcastle Disease Virus (NDV) could establish persistent infection in a tumor cell line resulting in a steady anti-viral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations each in the fusion (F) and Hemagglutinin-Neuraminidase (HN) proteins, respectively. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also sheds light into the critical balance between the F and HN proteins required for efficient NDV infection and in avian pathogenicity.

  • Mapping of transcription termination within the S segment of SFTS phlebovirus facilitated the generation of NSs-deletant viruses. [PublishAheadOfPrint]

  • SFTS phlebovirus (severe fever with thrombocytopenia syndrome virus; SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral non-structural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3rrsquo; RACE we mapped the 3rrsquo; -end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3rrsquo; end of the N mRNA terminates upstream of a 5rrsquo; -GCCAGCC-3rrsquo; motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nt of the NSs ORF were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate it is possible to recover viruses lacking NSs, expressing just a 12 amino acid NSs peptide or viruses encoding eGFP or a NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies.

    Importance SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV was shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the non-structural protein (NSs) enables the virus to counteract the human innate antiviral defences and that NSs is one of the major determinants of virulence in infection. Therefore, utilising reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonise the mammalian IFN response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping transcriptional termination of two S-segment derived subgenomic messenger RNAs, which revealed that transcription termination occurs upstream of a 5rrsquo; -GCCAGCC-3rrsquo; motif present in the virus genomic S RNA.

  • Gender parity trends for invited speakers at four prominent virology conference series [PublishAheadOfPrint]

  • Scientific conferences are most beneficial to participants when they showcase significant new experimental developments, accurately summarize the current state of the field, and provide strong opportunities for collaborative networking. A top-notch slate of invited speakers, assembled by conference organizers or committees, is key to achieving these goals. The perceived underrepresentation of female speakers at prominent scientific meetings is currently a popular topic for discussion, but one that often lacks supportive data. We compiled the full rosters of invited speakers over the last 35 years for four prominent international virology conferences, the American Society for Virology Annual Meeting (ASV), the International Herpesvirus Workshop (IHW), the Positive-Strand RNA Virus Symposium (PSR), and the Gordon Research Conference on Viruses aamp; Cells (GRC). The rosters were cross-indexed by unique names, gender, year, and repeat invitations. When plotted as gender-dependent trends over time, all four conferences showed a clear proclivity for male-dominated invited speaker lists. Encouragingly, shifts toward parity are emerging within all units, but at different rates. Not surprisingly, both selection of a larger percentage of first time participants and the presence of a woman on the speaker selection committee correlated with improved parity. Session chair information was also collected for the IHW and GRC. These visible positions also displayed a strong male dominance over time that is eroding slowly. We offer our personal interpretation of these data to aid future organizers achieve improved equity among the limited number of available positions for session moderators and invited speakers.

    IMPORTANCE Politicians and media members have a tendency to cite anecdotes as conclusions without any supporting data. This happens so frequently now, that a name for it has emerged: fake news. Good science proceeds otherwise. The under representation of women as invited speakers at international scientific conferences exemplifies a present-day discussion topic usually occurring without facts to support or refute the arguments. We now provide records profiling four prominent virology conferences over the years 1982 to 2017 with the intention that the trends and accompanying analyses of the gender parity of invited speakers may allow the ongoing discussions to be informed.

  • Time-sampled population sequencing reveals the interplay of selection and genetic drift in experimental evolution of Potato virus Y [PublishAheadOfPrint]

  • RNA viruses are one of the fastest evolving biological entities. Within their hosts, they exist as genetically diverse populations (i.e., viral mutant swarms), which are sculpted by different evolutionary mechanisms, such as mutation, natural selection and genetic drift, and also the interactions between genetic variants within the mutant swarms. To elucidate the mechanisms that modulate the population diversity of an important plant pathogenic virus, we performed evolution experiments with Potato virus Y (PVY) in potato genotypes that differ in their defense response against the virus. Using deep sequencing of small RNAs, we followed the temporal dynamics of standing and newly-generated variation in the evolving viral lineages. A time-sampled approach allowed us to: (i) reconstruct theoretical haplotypes in the starting population by using clustering of single nucleotide polymorphisms' trajectories and (ii) use quantitative population genetics approaches to estimate the contribution of selection and genetic drift, and their interplay, to the evolution of the virus. We detected imprints of strong selective sweeps and narrow genetic bottlenecks, followed by the shift in frequency of selected haplotypes. Comparison of patterns of viral evolution in differently susceptible host genotypes indicated possible diversifying evolution of PVY in the less susceptible host (efficient in the accumulation of salicylic acid).

    IMPORTANCE High diversity of within-host populations of RNA viruses is an important aspect of their biology, since they represent a reservoir of genetic variants, which can enable quick adaptation of viruses to changing environment. This study focuses on an important plant virus, Potato virus Y, and describes, at high resolution, temporal changes in the structure of viral populations within different potato genotypes. A novel and easy-to-implement computational approach was established to cluster single nucleotide polymorphisms into viral haplotypes from very short sequencing reads. During the experiment, a shift in the frequency of selected viral haplotypes was observed after a narrow genetic bottleneck, indicating an important role of the genetic drift in the evolution of the virus. On the other hand, a possible case of diversifying selection of the virus was observed in less susceptible host genotypes.

  • Novel cross-reactive monoclonal antibodies against Ebolavirus glycoproteins show protection in a murine challenge model [PublishAheadOfPrint]

  • Out of an estimated 31,100 cases since its discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (~11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species and the mechanism of these monoclonal antibody therapeutics are still not understood in detail. Here we generated a panel of eight murine monoclonal antibodies utilizing a prime boost DNA vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by an infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these mAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.

    IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans and our limited data indicates that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

  • Comprehensive cross-clade characterization of antibody-mediated recognition, complement-mediated lysis and cell-mediated cytotoxicity of HIV-1 envelope specific antibodies towards the eradication of the HIV-1 reservoir [PublishAheadOfPrint]

  • Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. Here, we determined the ability of twelve well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1, belonging to clades A, B, C and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro. We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, PGT145) are best at recognizing HIV-1 infected CD4 T cells. However, only PG9, PG16 and their combinations with other bnAbs, sufficiently induced the elimination of HIV-1 infected CD4 T cells by ADCML, ADCC or both. Notably, CD4 binding site antibodies VRC01, 3BNC117 and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared towards the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we detail a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics towards the development of a cure for HIV/AIDS.

    Importance: Several antibodies capable of neutralizing the majority of circulating HIV-1 viruses have been identified to-date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure.

  • Guanylate-binding protein 1 inhibits nuclear delivery of Kaposi's sarcoma-associated herpesvirus virions by disrupting formation of actin filament [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gamma-herpesvirus which establishes persistent lifelong infection in host cells. In order to establish a successful infection, KSHV has evolved numerous immune-evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during KSHV primary infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. Guanylate-binding protein 1 (GBP1) is one of the interferon stimulated genes and exerts antiviral functions on several RNA viruses, however its function in DNA virus infection is less well understood. In this study, we found that KSHV infection could increase both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-B pathway. Overexpression of GBP1 significantly inhibited KSHV infection while knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filament. Finally, we demonstrated that the replication and transcription activator (RTA) could promote the degradation of GBP1 protein through a proteasome pathway. Taken together, these results have provided new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity and suggests the critical role of RTA in the evasion of the innate immune response during the primary infection of KSHV.

    IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting nuclear delivery of KSHV virions via the disruption of actin filament. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome mediated pathway. Taken together, our results have elucidated a novel mechanism of GBP1 anti-KSHV activity and emphasized the critical role of RTA in KSHV evasion of the host immune system during primary infection.

  • Voltage-dependent anion channel 1 interacts with ribonucleoprotein complexes to enhance infectious bursal disease virus polymerase activity [PublishAheadOfPrint]

  • Infectious bursal disease virus (IBDV) is a double-stranded RNA virus. Segment A contains two overlapping open reading frames (ORFs), which encode viral proteins VP2, VP3, VP4, and VP5. Segment B contains one ORF and encodes the viral RNA-dependent RNA polymerase, VP1. IBDV ribonucleoprotein complexes are composed of VP1, VP3, and dsRNA and play a critical role in mediating viral replication and transcription during virus life cycles. Here, we identified a cellular factor, VDAC1, which was upregulated during IBDV infection and found to mediate IBDV polymerase activity. VDAC1 senses IBDV infection by interacting with viral proteins VP1 and VP3. This association is caused by RNA bridging and all three proteins co-localise in the cytoplasm. Furthermore, small interfering RNA (siRNA)-mediated downregulation of VDAC1 resulted in a reduction in viral polymerase activity and a subsequent decrease in viral yield. Moreover, overexpression of VDAC1 enhances IBDV polymerase activity. We also found that viral protein VP3 can replace segment A to execute polymerase activity. A previous study showed that mutations in the C-terminus of VP3 directly influence the formation of VP1nndash;VP3 complexes. Our immunoprecipitation experiments demonstrated that proteinnndash;protein interactions between VDAC1 and VP3 and between VDAC1 and VP1 play a role in stabilising the interaction between VP3 and VP1, further promoting IBDV polymerase activity.

    IMPORTANCE The cellular factor VDAC1 controls the entry and exit of mitochondrial metabolites and plays a pivotal role during intrinsic apoptosis by mediating the release of many apoptogenic molecules. Here, we identify a novel role of VDAC1, showing that VDAC1 interacts with IBDV ribonucleoproteins (RNPs) and facilitates IBDV replication by enhancing IBDV polymerase activity through its ability to stabilise interactions in RNP complexes. To our knowledge, this is the first report that VDAC1 is specifically involved in regulating IBDV RNA polymerase activity, providing novel insight into virusnndash;host interactions.

  • Evasion of the STING DNA sensing pathway by the VP11/12 of herpes simplex virus type 1 [PublishAheadOfPrint]

  • The STimulator of INterferon Genes (STING) is a broad antimicrobial factor that restricts HSV by activating type I interferon and pro-inflammatory responses upon sensing of foreign DNA. UL46 is one of the most abundant tegument proteins of HSV-1 but a well-established function has yet to be found. We found that the HSV-1 UL46 protein interacts with and co-localizes with STING. A UL46 virus displayed growth defects and activated innate immunity but both effects were alleviated in STING-knockdown cells. UL46 was also required for the inhibition of the 2rrsquo; 3rrsquo; -cGAMP-dependent immune responses during infection. In cells expressing UL46, out of the context of the infection, innate immunity to a ICP0 virus was largely compromised and that permitted ICP0-deficient mutants to replicate. The UL46-expressing cell lines rescued also the defects of the UL46 virus and enhanced wild type virus infection. The UL46-expressing cell lines did not activate interferon-stimulated gene transcription (ISGs) following treatment with the non-canonical cyclic dinucleotide 2rrsquo; 3rrsquo; -cGAMP, suggesting that the STING pathway may be compromised. Indeed, we found that both proteins STING and IFI16 were eliminated in cells constitutively expressing UL46 and the accumulation of their transcripts was blocked. Finally, we demonstrated that UL46 via its N-terminus binds to STING and via its C-terminus to TBK1. These interactions appear to modulate the functions of STING during HSV-1 infection. Taken together, our studies describe a novel function for one of the least studied proteins of HSV, the tegument protein UL46, and that function involves the evasion of foreign DNA sensing pathways.

    IMPORTANCE Herpes simplex virus-1 (HSV-1) afflicts 80% of the population worldwide causing various diseases. After initial infection, the virus establishes latent reservoirs in sensory neurons and persists lifelong. Here, we describe novel interactions between HSV-1 and the DNA sensor STING. We found that: (i) the tegument protein of HSV-1 UL46 interacts with and colocalizes with STING; (ii) UL46 expressed out of the context of the infection blocks type I interferon triggered by STING stimuli, through the elimination of STING and of the interferon inducible protein 16 (IFI16); (iii) a UL46 virus displayed growth defects, which were rescued in STING-knockdown cells; (iv) the UL46 virus failed to block innate immunity triggered by ligands of STING such as 2rrsquo; 3rrsquo; -cGAMP and also activated IFN-bbeta; and ISGs expression; (v) UL46 binds to both STING and TBK1 through different domains. We conclude that UL46 counteracts the actions of STING during HSV-1 infection.

  • Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector Against Dengue Virus in Rhesus Monkeys [PublishAheadOfPrint]

  • Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed the non-structural protein 5 (NS5) while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 x 105 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following the high dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against Dengue virus infection.

    IMPORTANCE Continuing study of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here, we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against dengue virus 2 (DENV2) infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high dose DENV2 challenge 19 weeks post-vaccination. Furthermore, one of these vaccinated monkeys appeared to be protected against the acquisition of DENV2 infection on the basis of undetectable viral loads and the lack of an anamnestic antibody response. These findings underscore the potential utility of recombinant herpesviruses as vaccine vectors.

  • Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane Budding Activity [PublishAheadOfPrint]

  • Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher oligomers may facilitate membrane budding. To gain functional insights, we here employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane budding activity. Three six-alanine-scan (6A) block mutants, locating at strategic oligomeric positions, were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the two others display amino acids locating within two distal surface-exposed aalpha;-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating the asparagine 138 (N138) into several non-conservative amino acids. Cellular localization combined with dimerization and co-immunopurification assays, performed under various denaturing conditions, revealed that all 6A-mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiency in relocating CDV nucleocapsid proteins to the cell periphery and virus-like particles (VLP) production. Conversely, all N138 M mutants remained capable of self-assembly, although to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that M-N138 variants exhibiting most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provided mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane budding activity.

    IMPORTANCE Despite availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that post-exposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here, we combined structure-guided mutagenesis with cellular, biochemical and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provided evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher oligomers necessary to promote plasma membrane budding activity.

  • Zika virus hijacks stress granule proteins and modulates the host stress response [PublishAheadOfPrint]

  • Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barreeacute; syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2aalpha; and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3 and NS4A interfered with SG formation. Some but not all flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR and Caprin-1, but not TIA-1 reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.

    IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccine and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection and therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Herein, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shut-off, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

  • Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation to Promote Lytic Replication [PublishAheadOfPrint]

  • N6-adenosine methylation (m6A) is the most common post-transcriptional RNA modification in mammalian cells. We found that most transcripts encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) undergo m6A modification. The levels of m6A-modified mRNAs increase substantially upon stimulation for lytic replication. Blocking m6A inhibits splicing of the pre-mRNA encoding replication transcription activator (RTA), a key KSHV lytic switch protein, and halts viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induces m6A and enhances its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication.

    IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of KS tumors. Previous studies suggest that KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV-encoded mRNAs undergo m6A modification, which represents a new mechanism at the post-transcriptional level in the control of viral replication.

  • Covalent linkage of HIV-1 trimers to synthetic liposomes elicits improved B cell and antibody responses [PublishAheadOfPrint]

  • We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimer via their C-terminal poly-histidine tails to nickel-lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remains over the potentially longer term in vivo instability of non-covalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the poly-histidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, but it also completely eliminated antibodies directed to the C-terminal His-tag located at the "bottom" of the spike. In contrast, soluble and non-covalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes at high-density array displays multiple advantages in vitro and in vivo.

    IMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surface mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a non-covalent linkage and improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have an improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-baring trimers via non-covalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a non-neutralizing determinant located at the bottom of the trimer.

  • Identification of IL-27/IL-27RA as a critical immune axis for in vivo HIV control [PublishAheadOfPrint]

  • Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray able to measure ggt;600 plasma proteins involved in cell-to-cell communication was used to measure plasma protein profiles in 96 HIV-infected, treatment-naïve individuals with high (ggt;50,000) or low (llt;10,000 HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble IL-27 are significantly elevated in individuals with high plasma viremia (pllt;0.0001) and are positively correlated with proviral HIV-DNA copy numbers in PBMC (Rho=0.4011 p=0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T cell responses and directly with plasma levels of molecules involved in Wnt/bbeta;-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor (IL27RA) in PBMC correlated directly with both, plasma viral load (Rho=0.3531 p=0.0218) and the proviral copy number in the peripheral blood, as an indirect measure of partial viral reservoir (Rho=0.4580 p=0.0030). These results were validated in unrelated cohorts of early-infected subjects as well as subjects before and after initiation of antiretroviral treatment, and identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC.

    IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins and the blood is a key conduit for transporting such factors. Investigating the communication factors promoting effective immune responses and having potentially antiviral functions against HIV using a novel focused nndash;omics approach ("Communicome") has the potential to significantly improve our knowledge on effective host immunity and accelerate the HIV cure agenda. Including 140 subjects with variable viral loads and measuring the plasma levels of ggt;600 soluble proteins, our data highlight the importance of Th17 cells and Wnt/bbeta;-catenin Signaling in HV control and especially identify IL-27/IL-27RA axis as a predictor of plasma viral load and proviral copy number in the peripheral blood. These data may provide important guidance to therapeutic approaches in the HIV cure agenda.

  • HDM2 promotes NEDDylation of HBV HBx to enhance its stability and function [PublishAheadOfPrint]

  • Hepatitis B virus-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrated that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitination-mediated degradation. Consistently, analysis of 160 HCC patient specimens indicated that the amount of HDM2 protein correlates with HBx protein level. We identified that HBx K91 and K95 as the key HBx NEDDylation sites and observed that the NEDDylation-deficient HBx has shorter half-life. We generated Huh7 cell lines which ectopically express wild-type and NEDDylation deficient HBx, and found that NEDDylation-deficient HBx showed less chromatin localization and less DDB1 binding. Consistently, the expression of HBx-regulated genes (IL-8, MMP9 and YAP) and the HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing WT HBx than that in cells expressing mutant HBx. In addition, HBx-expressing cells proliferated faster than control and mutant HBx-expressing cells. We also showed that the ability of WT HBx-expressing cells to form tumor in nude mice was significantly higher than that of mutant HBx-expressing cells. In conclusion, we revealed that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability and chromatin localization which in turn favors HBx-dependent transcriptional regulation, cell proliferation and promoting HBV-driven tumor growth.

    IMPORTANCE HBV HBx protein plays a critical role in viral replication and hepatocarcinogenesis. However, the regulation of HBx stability is not well understood. We found that HBx is modified by NEDD8 and that the HDM2 E3 ligase promotes HBx NEDDylation to enhance HBx stability by inhibiting its ubiquitination. We provide a new evidence to show the positive correlation between HDM2 and HBx in clinical HCC samples. We also identified the major Neddylation sites on HBx. Our studies indicate that the defective NEDDylation of HBx negatively affects its ability to activate transcription of downstream genes, promote cell proliferation and tumor growth in vivo. Taken together, we revealed a novel posttranslational modification of HBx by HDM2 which regulates its stability, subcellular localization and functions. These findings indicate that HDM2 is an important regulator on HBx and a potential diagnosis/therapeutic marker for HBV-associated HCC.

  • Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 peptide in Marburg virus [PublishAheadOfPrint]

  • Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNP undergo a transition from RNA-bound form to template-free form, to open access for the interaction between viral polymerase with RNA template. In filovirus, this transition is regulated by the VP35 and other viral components. To further understand the dynamic process of filovirus RNP formation, here we report the structure of MARV NPcore, both in apo form and VP35 peptide chaperoned form. These structures reveal a typical bi-lobed structure, with a positive charged RNA binding groove between two lobes. In the apo form, the MARV NP existed as an interesting hexameric state formed by the hydrophobic interaction within long helix of the NP C-terminal region, which shows high structural flexibility among filovirus and may imply critical function during RNP formation. Moreover, the VP35 peptide chaperoned NPcore keeps in monomeric state and completely lost its affinity for ssRNA. The structural comparison reveals that RNA binding groove undergoes a transition from close state to open state, chaperoned by VP35 peptide, thus prevent the interaction for viral RNA. Our investigation provides a great structural insight into the filovirus RNP working mechanism, and may support the development of antiviral therapies targeting the RNP formation of filovirus.

    Importance Marburg virus is one of the most dangerous viruses with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 people. NP is one of the most essential proteins by encapsidating and protecting the whole virus genome contemporary with self-assembly oligomerization. Here we reported the structures of MARV NPcore in two different forms. In MARV NP apo form, we identify an interesting hexamer formed by hydrophobic interaction within a long helix, which is highly conservative and flexible among filovirus and may indicate its critical function during the virus RNP formation. Moreover, the structural comparison with the NP-VP35 peptide complex reveals structural transition chaperoned by VP35, in which RNA binding groove undergoes a transition from close state to open state. Finally, we discussed the high conservation and critical role of the VP35 binding pocket and its potential use for therapeutic development.

  • Seneca Valley Virus suppresses host type I interferon production by targeting adaptor proteins MAVS, TRIF, and TANK for cleavage [PublishAheadOfPrint]

  • Seneca Valley Virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anti-cancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial anti-viral signaling (MAVS), Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), and TRAF family member-associated NF-B activator (TANK) via viral 3Cpro. SVV 3Cpro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost their ability to regulate PRR-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-B activation. SVV was also found to be sensitive to IFNbbeta;. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules.

    IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the picornavirus genus, is an oncolytic virus that exerts potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. Here, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3Cpro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for the TLR3- and RLR-mediated signaling pathway. We also found that SVV is sensitive to IFNbbeta;. These findings increase our understanding of the interaction between SVV and host innate immunity.

  • Drug modulators of B cell signaling pathways and Epstein-Barr virus lytic activation [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block BCR signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mTORC2 contributes to BCR-mediated lytic induction and that FKBP12 binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMC) and that activation can be inhibited by ibrutinib or idelalisib.

    IMPORTANCE EBV establishes viral latency in B cells. Activation of the B-cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression, but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR-activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR-inhibitors.

  • The SAT protein of porcine parvovirus accelerates viral spreading through irreversible ER stress induction [PublishAheadOfPrint]

  • The SAT protein of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER) and SAT deletion induces "slow spreading" phenotype. The in vitro comparison of the wild type Kresse strain and its SATnndash; knockout mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SATnndash; virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences were detected in the quantity and the localization of CHOP, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of irreversible ER stress induction in PT cell necrosis and the viral egress was confirmed by treatment of infected cells by ER stress inducing chemicals (MG132, DTT and Thapsigargin) that accelerated the egress and spreading both the wild type and the SATnndash; viruses. UV stress induction had no beneficial effect to PPV infectionunderscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a co-expression experiment.

    IMPORTANCE SATp is encoded in an alternative ORF of the PPV genome. Earlier we showed that SATp of the attenuated PPV-NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that "slow spreading" is a general feature of the SATnndash; PPV viruses and is the consequence of prolonged cell integrity. PPV infection induced ER stress in the infected cells regardless of SATp presence, as demonstrated by the morphological changes of the ER, and expression of the stress response proteins XBP1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated the cell death during infection as shown by the higher expression rate and the alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by the treatment of the infected cells with ER stress inducing chemicals.

  • The number of target molecules of the amplification step limits accuracy and sensitivity in ultra deep sequencing viral population studies [PublishAheadOfPrint]

  • The invention of next generation sequencing techniques (NGS) marked the coming of a new era in the detection of genetic diversity of intra-host viral populations. A good understanding of the genetic structure of these populations first requires being able to identify the different isolates or variants, and second to accurately quantify them. However, the initial amplification step of NGS studies can impose potential quantitative biases modifying the variant relative frequencies. In particular, the number of target molecules (NTM) used during the amplification step is vastly overlooked, though of primary importance as it sets the limit of the accuracy and the sensitivity of the sequencing procedure. In the present article, we investigated quantitative biases in the NGS study of populations of a multipartite ssDNA virus at different steps of the procedure. We studied 20 independent populations of the ssDNA Faba Bean Necrotic Stunt Virus (FBNSV) virus in two host plants, Faba bean and Medicago. The FBNSV is a multipartite virus composed of eight genomic segments, whose specific and host-dependent relative frequencies are defined as the "genome formula". Our results show significant distortion of the FBNSV genome formula after the amplification and the sequencing steps. We also quantified the genetic bottleneck occurring at the amplification step by documenting the NTM of two genomic segments of the FBNSV. We argue that the NTM must be documented and carefully considered when interpreting the sensitivity and accuracy of NGS studies.

    Importance The advent of next generation sequencing techniques (NGS) now enables studying the genetic diversity of viral populations. A good understanding of the genetic structure of these populations first requires being able to identify the different isolates or variants, and second to accurately quantify them. Prior to sequencing, viral genomes need to be amplified, a step that potentially imposes quantitative biases and modifies the viral population structure. In particular, the number of target molecules (NTM) used during the amplification step is of primary importance as it sets the limit of the accuracy and the sensitivity of the sequencing procedure. In this work, we used 20 replicated populations of the multipartite Faba Bean Necrotic Stunt Virus (FBNSV) to estimate the various limitations of ultra deep sequencing studies performed on intra-host viral populations. We report quantitative biases during Rolling Circle Amplification and the NTM of two genomic segments of the FBNSV.

  • Human papillomavirus major capsid protein L1 remains associated with the incoming viral genome throughout the entry process [PublishAheadOfPrint]

  • During infectious entry, acidification within the endosome triggers uncoating of the HPV capsid whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans-Golgi network (TGN). Following the onset of mitosis, HPV-harboring transport vesicles bud from the TGN followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggests that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity and L2 protein has multiple domains capable of interacting with L1 capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full-length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and require assembly into capsomeres, we propose that L1 protein is present in form of pentamers. Furthermore, we provide evidence that L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking.

    IMPORTANCE After internalization, the non-enveloped human papillomavirus virion uncoats in the endosome whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggests that some L1 protein may accompany the viral genome beyond the endosomal compartment. Herein, we demonstrate that conformationally intact L1 protein, likely still arranged as capsomeres, remains associated with the incoming viral genome throughout mitosis and transiently resides in the nucleus until after the viral DNA is released from the transport vesicle.

  • Discovery and mechanistic study of benzamide derivatives that modulate hepatitis B virus capsid assembly [PublishAheadOfPrint]

  • Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein, but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the "HAP" pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated slower in native agarose gel electrophoresis from A36V mutant core protein. Moreover, we showed that assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence HBV core protein is a dominant antiviral target that may suppress the selection of drug resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV.

    IMPORTANCE HBV core protein plays essential roles in many steps of viral replication cycle. In addition to packaging viral pregenomic (pg) RNA and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating and cccDNA formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. Discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported herein, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B.

  • Ruxolitinib and polycation combination treatment overcomes multiple mechanisms of resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus [PublishAheadOfPrint]

  • Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of the resistance are still unclear. JAK 1/2 inhibitors (such as ruxolitinib and JAK Inhibitor 1) strongly stimulate VSV replication and oncolysis in all resistant cell lines, but only partially improve susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication, rather than on receptor specificity, with several ubiquitously expressed cell-surface molecules to play a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDACs. Our data show a dramatically weaker attachment of VSV to HPAF-II, the most resistant human PDAC cell line. Although sequence analysis of LDLR mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels, but did not improve VSV attachment or LDL uptake in HPAF-II. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and polybrene or DEAE-dextran successfully broke the resistance of HPAF-II to VSV by simultaneously improving VSV attachment and replication.

    IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC). Although VSV is effective against most PDACs, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDACs. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (inhibits antiviral signaling) successfully broke the resistance of HPAF-II to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple combination approach could be used in the future to treat PDAC tumors highly resistant to OV therapy.

  • A Critical Role for Monocytes/Macrophages in the Rapid Progression to AIDS in Pediatric SIV-Infected Rhesus Macaques [PublishAheadOfPrint]

  • Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV, SIV) respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4+ T cells in intestinal tissues are major primary target cells for SIV/HIV infection and massive depletion of these cells is considered a major cause of immunodeficiency. Monocytes and macrophages are important cells of innate immunity and also are targets of HIV/SIV infection. We reported previously that high peripheral blood monocyte turnover rate was predictive for the onset of disease progression to AIDS in SIV-infected adult macaques. The purpose of this study was to determine if earlier or higher infection of monocytes/macrophages contributes to the more rapid progression to AIDS in infants. We observed that uninfected infant rhesus macaques exhibited higher physiologic baseline monocyte turnover than adults. Early after SIV infection, the monocyte turnover further increased and remained high during progression to AIDS. A high percentage of terminal deoxynucleotidyl transferase dUTP nick-end label-(TUNEL)-positive macrophages in the lymph nodes (LNs) and intestine corresponded with an increasing number of macrophages derived from circulating monocytes (BrdU+ CD163+), suggesting that the increased blood monocyte turnover was required to rapidly replenish destroyed tissue macrophages. Immunofluorescence analysis further demonstrated that macrophages were a significant portion of the virus-producing cells found in LNs, intestinal tissues, and lungs. The higher baseline monocyte turnover in infant macaques and subsequent macrophage damage by SIV infection may help explain the basis of more rapid disease progression to AIDS in infants.

    IMPORTANCE HIV infection progresses much more rapidly in pediatric cases than in adults, however the mechanism for this difference is unclear. Using the rhesus macaque model, this work was performed to address why infants infected with SIV progress more quickly to AIDS than do adults. Earlier we reported that in adult rhesus macaques, increasing monocyte turnover reflected tissue macrophage damage by SIV and was predictive of terminal disease progression to AIDS. Here, we report that uninfected infant rhesus macaques exhibited a higher physiological baseline monocyte turnover rate than adults. Furthermore, once infected with SIV, infants displayed further increased monocyte turnover that may have facilitated the accelerated progression to AIDS in infected pediatric patients/. These results continue to support a role for monocytes and macrophages in the pathogenesis of SIV/HIV and begin to explain why infants are more prone to rapid disease progression.

  • Hepatitis C Virus Subverts Human Choline Kinase-{alpha} to Bridge PI4KIII{alpha} and NS5A and Upregulates PI4KIII{alpha} Activation, Thereby Promoting the Translocation of the Ternary Complex to the ER for Viral Replication [PublishAheadOfPrint]

  • In this study, we elucidated the mechanism by which human choline kinase-aalpha; (hCKaalpha;) interacts with nonstructural protein (NS)5A and phosphatidylinositol-4-kinase (PI4K)IIIaalpha;, the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKaalpha; activity positively modulated PI-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKaalpha;-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIaalpha;-specific inhibitor. hCKaalpha; colocalized with NS5A and PI4KIIIaalpha; or PI4P, NS5A expression increased hCKaalpha; and PI4KIIIaalpha; colocalization, and hCKaalpha; formed a ternary complex with PI4KIIIaalpha; and NS5A, supporting the functional interplay of hCKaalpha; with PI4KIIIaalpha; and NS5A. PI4KIIIaalpha; inactivation by AL-9 or hCKaalpha; inactivation by CK37, a specific hCKaalpha; inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKaalpha; knockdown or inactivation inhibited PI4KIIIaalpha;-NS5A binding. In an in vitro PI4KIIIaalpha; activity assay, hCKaalpha; activity slightly increased PI4KIIIaalpha; basal activity but greatly augmented NS5A-induced PI4KIIIaalpha; activity, supporting the essential role of ternary complex formation in robust PI4KIIIaalpha; activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKaalpha;-R (but not the D288A mutant) restored PI4KIIIaalpha; and NS5A translocation to the ER in hCKaalpha; stable knockdown cells. Furthermore, active PI4KIIIaalpha; overexpression restored PI4P production, PI4KIIIaalpha; and NS5A translocation to the ER and viral replication in CK37-treated cells. Based on our results, hCKaalpha; functions as an indispensable regulator that bridges PI4KIIIaalpha; and NS5A and potentiates NS5A-stimulated PI4KIIIaalpha; activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.

    IMPORTANCE The mechanisms by which hCKaalpha; activity modulates the transport of the hCKaalpha;-NS5A complex to the ER are not understood. In the present study, we investigated how hCKaalpha; interacts with PI4KIIIaalpha;, a key element that maintains the integrity of the "membranous web" structure, and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKaalpha; to bridge PI4KIIIaalpha; and NS5A together via hCKaalpha; activity to form a ternary complex, which then stimulates PI4KIIIaalpha; activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for viral replication complex assembly and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKaalpha; in the recruitment of PI4KIIIaalpha; to NS5A and NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIaalpha; activation on PI4KIIIaalpha; and NS5A targeting to the PI4P-enriched membrane for viral replication complex formation.

  • Vimentin modulates infectious internalisation of HPV16 pseudovirions [PublishAheadOfPrint]

  • Human papillomavirus (HPV) is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells is controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase and subsequent transfer of the virus to an as yet unidentified high-affinity receptor. In line with this model we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were non-permissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pull-down assays followed by mass spectrometry analysis showed that furin pre-cleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and -uncleaved HPV16-PsVs co-localised with surface expressed vimentin on pgsD677, HeLa, HaCaT and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by siRNA knockdown in HeLa and NIKS cells significantly increased HPV16-PsVs infectious internalisation, while overexpression of vimentin had the opposite effect.

    The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for novel antiviral drug design preventing HPV internalisation into epithelial cells.

    IMPORTANCE Despite being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalisation are poorly understood. We herein describe the identification of surface expressed vimentin as a novel molecule not previously implicated in the infectious internalisation of HPV16. Contrary to our expectations, vimentin was found not to act as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalisation of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

  • Rapid Engineering for Vaccine and Challenge Viruses against Foot-and-Mouth Disease [PublishAheadOfPrint]

  • There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intra-typic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-coding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT 1, SAT 2, and SAT 3 serotypes. The seven serotype viruses were rescued successfully. Each chimeric FMDV with replacing P1 showed its serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Pigs vaccinated with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for customized vaccine with challenge tool to protect against epizootic disease from specific serotypes or subtypes of FMDV.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) causes significant economic losses. For the vaccine preparation, the selection of vaccine strains was complicated by its high antigenic variation. In the present study, we suggested that an effective strategy can be rapidly prepare and evaluate a mass-producing customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of seven representative serotype viruses. These chimeric viruses generally replicated readily in cell culture and had the similar particle size as the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses against all serotypes, especially for FMD-free countries which have prohibited import of FMDVs.

  • Interactions Between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly [PublishAheadOfPrint]

  • Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious virus-like particles and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA:Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.

    IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we showed that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

  • Epigenetic metabolite acetate inhibits class I/II histone deacetylases, promotes histone acetylation and increases HIV-1 integration in CD4+ T cells [PublishAheadOfPrint]

  • In this study, we investigated the effect of acetate, the most concentrated short-chain fatty acid (SCFA) in the gut and bloodstream, on the susceptibility of primary human CD4+ T cells to HIV-1 infection. We report that HIV-1 replication is increased in CD3/CD28-costimulated CD4+ T cells upon acetate treatment. This enhancing effect correlates with an increased expression of the early activation marker CD69 and impaired class I/II histone deacetylase (HDAC) activity. In addition, acetate enhances acetylation of histones H3 and H4 and augments HIV-1 integration in the genome of CD4+ T cells. Thus, we propose that upon antigen presentation, acetate influences class I/II HDAC activity that transforms condensed chromatin into a more relaxed structure. This event leads to a higher level of viral integration and an enhanced HIV-1 production. In line with previous studies showing reactivation of latent HIV-1 by SCFAs, we provide evidence herein that acetate can also increase susceptibility of primary human CD4+ T cells to productive HIV-1 infection.

    IMPORTANCE Alterations in the fecal microbiota and intestinal epithelial damages involved in the gastrointestinal disorder associated with HIV-1 infection result in microbial translocation that leads to disease progression and virus-related comorbidities. Indeed, notably via production of short-chain fatty acids (SCFAs), bacteria migrating from the lumen to the intestinal mucosa could influence HIV-1 replication by causing epigenetic regulatory mechanisms such as histone acetylation. We demonstrate that acetate enhances virus production in primary human CD4+ T cells. Moreover, we report that acetate impairs class I/II histone deacetylase activity and increases integration of HIV-1 DNA into the host genome. Therefore, it can be postulated that bacterial metabolites such as acetate modulate HIV-1-mediated disease progression.

  • Virological control by the CD4-binding site antibody N6 in SHIV-infected rhesus monkeys [PublishAheadOfPrint]

  • Passive immunotherapies against HIV-1 will most likely require broadly neutralizing antibodies (bnAb) with maximum breadth and potency to assure therapeutic efficacy. Recently, the novel CD4 binding site antibody N6 demonstrated extraordinary neutralization breadth and potency against large panels of cross clade pseudoviruses. We evaluated the in-vivo antiviral activity of N6-LS, alone or in combination with the established V3-glycan antibody PGT121, in chronically SHIV-SF162P3 infected macaques. A single dose of N6-LS suppressed plasma viral loads in 4 out of 5 animals at day 7 (mean 1.1 log10 RNA copies/ml reduction), while the combination of both antibodies suppressed all animals (mean 0.92 log10 RNA copies/ml reduction). Interestingly, the combination of both antibodies had no additive antiviral effect, compared to a single dose of PGT121, potentially reflecting the nearly 10-fold higher potency of PGT121 against this SHIV. Viral rebound occurred in the majority of suppressed animals and was linked to declining plasma bnAb levels over time. In addition to the effect on plasma viremia, bnAb administration resulted in significantly reduced proviral DNA levels in PBMCs after 2 weeks and in lymphnode cells after 10 weeks. Autologous NAb responses and SIV/SHIV specific CD8+ T-cell responses were not significantly enhanced in the bnAb treated animals compared to control animals, arguing against their contribution to the viral effects observed. These results confirm the robust antiviral activity of N6-LS in-vivo, supporting the further clinical development of this antibody.

    IMPORTANCE Monocloncal antibodies (mAbs) are being considered for passive immunotherapies of HIV-1 infection. A critical requirement for such strategies is the identification of mAbs that recognize the diversity of variants within circulating but also reservoir viruses and mAb combinations might be needed to achieve this goal. This study evaluates the novel bnAb N6-LS, that has superior in-vitro antiviral characteristics, alone or in combination with the bnAb PGT121 in rhesus macauqes that are chronically infected with chimeric simian-human immunodeficiency virus (SHIV). The results demonstrate that N6-LS potently suppressed plasma viral loads in the majority of animals but that the combination with PGT121 was not superior than PGT121 alone in delaying time to viral rebound or reducing PBMC or lymphnode cell proviral DNA levels. The occurrence of viral escape variants in an N6-LS mono-treated animal, however, argues for the critical need to maximize breadth and anti-viral efficacy by combining bnAbs for therapeutic indications.

  • Ebola Virus Delta Peptide is a Viroporin [PublishAheadOfPrint]

  • The Ebola virus (EBOV) genome encodes for a partly conserved, 40-residue, nonstructural polypeptide, called the delta peptide, which is produced in abundance during Ebola virus disease. The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey and human origin, increase ion permeability across confluent cell monolayers and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin, and may be a novel, targetable aspect of Ebola virus disease pathology.

    Importance During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved non-structural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology, and a possible target for novel countermeasures.

  • Functional consequences of RNA 5 terminal deletions on coxsackievirus B3 RNA replication and ribonucleoprotein complex formation [PublishAheadOfPrint]

  • Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5rrsquo; terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5rrsquo; stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo on coxsackievirus B3 biology. Using cell-free extracts from HeLa cells as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5rrsquo; deletions ranging from 7 to 49 nucleotides can be translated nearly as efficiently as those of wild-type virus. However, these 5rrsquo; deletions greatly reduced the synthesis of viral RNA in vitro, which was only detected for the 7 and 21 nucleotide deletions. Since 5rrsquo; stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of host cell protein PCBP2 as well as viral protein 3CDpro to deleted positive-strand RNAs corresponding to the 5rrsquo; end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CDpro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5rrsquo; deleted viral genomes - a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.

    IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one third of idiopathic, dilated cardiomyopathies. Terminal deletions at the 5rrsquo; end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5rrsquo; terminal sequence deletions are able to direct the synthesis of viral proteins but not genomic RNAs in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions, but that the impaired RNA synthesis associated with terminally deleted viruses could be due to destabilization of the ribonucleoprotein complexes formed.

  • Mimic Phosphorylation of a {beta}C1 Encoded by TYLCCNB Impairs its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant [PublishAheadOfPrint]

  • Phosphorylation of the bbeta;C1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-bbeta;C1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana. However, how phosphorylation of TYLCCNB-bbeta;C1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-bbeta;C1 that are phosphorylated by SnRK1. The effects of TYLCCNB-bbeta;C1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation- mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-bbeta;C1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-bbeta;C1 to reverse transcriptional gene silencing and suppress post-transcriptional gene silencing, and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-bbeta;C1 had no impact on its protein stability, subcellular localization or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-bbeta;C1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation on how SnRK1 functions as a host defense factor.

    IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein bbeta;C1 (TYLCCNB-bbeta;C1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we showed that mimicking phosphorylation of TYLCCNB-bbeta;C1 weakens its ability to reverse transcriptional gene silencing, suppress post-transcriptional gene silencing and interact with N. benthamiana ASYMMETRIC LEAVES 1. To the best of our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-bbeta;C1 on its pathogenic functions as both a VSR and a symptom determinant, and to provide a mechanistic explanation on how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.

  • Structure of Simian Immunodeficiency Virus Envelope Spikes bound with CD4 and Monoclonal Antibody 36D5 [PublishAheadOfPrint]

  • The HIV-1/SIV envelope spike (Env) mediates the viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the co-receptor binding site and is a target for neutralizing antibodies. We have used cryoelectron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody 36D5 to gp120 of the SIV Env. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest the antibody exerts its neutralization effect by blocking the co-receptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 with SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 for gp120 in the intact Env consistent with CD4 induced changes in the conformation of gp120 and the antibody-binding site. Binding by MAb 36D5 does not alter substantially the proportions of the two CD4 bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations indicating that the V3 base serves as a pivot point during the transition between these two states.

    IMPORTANCE Glycoprotein spikes located on the surface of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that mask the epitope conformation. Using SIV virions whose spikes had been "decorated" with the primary cellular receptor, CD4, and an antibody, 36D5, to part of the co-receptor-binding site, we visualize multiple conformations trapped by the rapid freezing step which were separated using statistical analysis. Our results show that the CD4 induced conformational dynamics in the spike enhances binding of the antibody.

  • A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations [PublishAheadOfPrint]

  • Understanding the diversity and consequences of viruses present in honey bees is critical to maintain pollinator health and manage the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as Deformed wing virus. Previous genomic studies have focused on colonies suffering from Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites, but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or been selected for resistance to Varroa. This revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two Rhabdoviruses were present in three geographically diverse locations, and were also in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management.

    IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral meta-transcriptomics on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses, and will ultimately contribute to improved disease management in our most important agricultural pollinator.

  • Crosslinking of a CD4 Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques [PublishAheadOfPrint]

  • Evaluation of the epitope specificities, location (systemic, mucosal) and effector function of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitude, epitope specificity, avidity and function of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4 mimetic miniprotein (gp140-M64U1) in rhesus macaques. Crosslinking of gp140 Env with M64U1 resulted in an earlier increase in both the magnitude and avidity of the IgG binding response compared to Env protein alone. Notably, binding IgG responses at an early time point correlated with Antibody Dependent Cellular Cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the crosslinked Env group compared to the Env group alone. In addition, the crosslinked Env group developed higher IgG responses against a linear epitope in the C1 gp120 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by crosslinking gp140 with the CD4 mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses that correlated with the rise of subsequent antibody-mediated antiviral functions.

    IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and the immune correlates analysis of this trial revealed a role for binding antibodies and antibody Fc mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that Env structural modifications that aim to mimic the CD4 bound conformation can result in earlier antibody elicitation, altered epitope specificity and increased antiviral function post immunization.

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