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Table of Contents for this page:

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

    JVI Current Issue

  • Conservation and Divergence of the I-Domain Inserted into the Ubiquitous HK97 Coat Protein Fold in P22-Like Bacteriophages [Structure and Assembly]

  • Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel bbeta;-barrels. Nevertheless, significant structural differences occur in loops connecting the bbeta;-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions.

    IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded bbeta;-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages.

  • Editorial Board [Masthead]

  • PPAR-{gamma} in Macrophages Limits Pulmonary Inflammation and Promotes Host Recovery following Respiratory Viral Infection [Pathogenesis and Immunity]

  • Alveolar macrophages (AM) play pivotal roles in modulating host defense, pulmonary inflammation, and tissue injury following respiratory viral infections. However, the transcriptional regulation of AM function during respiratory viral infections is still largely undefined. Here we have screened the expression of 84 transcription factors in AM in response to influenza A virus (IAV) infection. We found that the transcription factor PPAR- was downregulated following IAV infection in AM through type I interferon (IFN)-dependent signaling. PPAR- expression in AM was critical for the suppression of exaggerated antiviral and inflammatory responses of AM following IAV and respiratory syncytial virus (RSV) infections. Myeloid PPAR- deficiency resulted in enhanced host morbidity and increased pulmonary inflammation following both IAV and RSV infections, suggesting that macrophage PPAR- is vital for restricting severe host disease development. Using approaches to selectively deplete recruiting monocytes, we demonstrate that PPAR- expression in resident AM is likely important in regulating host disease development. Furthermore, we show that PPAR- was critical for the expression of wound healing genes in AM. As such, myeloid PPAR- deficiency resulted in impaired inflammation resolution and defective tissue repair following IAV infection. Our data suggest a critical role of PPAR- expression in lung macrophages in the modulation of pulmonary inflammation, the development of acute host diseases, and the proper restoration of tissue homeostasis following respiratory viral infections.

    IMPORTANCE Respiratory viral infections, like IAV and respiratory syncytial virus (RSV) infections, impose great challenges to public health. Alveolar macrophages (AM) are lung-resident immune cells that play important roles in protecting the host against IAV and RSV infections. However, the underlying molecular mechanisms by which AM modulate host inflammation, disease development, and tissue recovery are not very well understood. Here we identify that PPAR- expression in AM is crucial to suppress pulmonary inflammation and diseases and to promote fast host recovery from IAV and RSV infections. Our data suggest that targeting macrophage PPAR- may be a promising therapeutic option in the future to suppress acute inflammation and simultaneously promote recovery from severe diseases associated with respiratory viral infections.

  • V1 and V2 Domains of HIV Envelope Contribute to CCR5 Antagonist Resistance [Vaccines and Antiviral Agents]

  • Vicriviroc (VCV) is a CCR5 antagonist that blocks the viral entry of CCR5-tropic (R5) virions by binding to and inducing a conformational change in the chemokine receptor. Clinical resistance to CCR5 antagonists occurs in two phases, competitive and noncompetitive stages. In this study, we analyzed two subjects, from a phase 2b VCV clinical trial, whose quasispecies contained R5 and dual-mixed virions at the earliest recorded time of virological failure (VF). Genotypic analysis of R5-tropic patient-derived envelope genes revealed significant changes in the V1/V2 coding domain and convergence toward a more homogenous sequence under VCV therapy. Additionally, a small population of baseline clones sharing similar V1/V2 and V3 domains with the predominant VF isolate was observed. These clones were denoted preresistant based on their genotype. Preresistant clones and chimeric clones containing V1/V2 regions isolated during VF displayed high 50% inhibitory concentration (IC50) values relative to those at baseline, consistent with early competitive resistance. Genotypic analysis of the dual-tropic clones also showed significant changes in the V1/V2 region, different from the resistant R5-tropic viruses. Our findings suggest that the V1/V2 domain plays a key role in the initial step of development of drug resistance.

    IMPORTANCE It is believed that each CCR5 antagonist-resistant isolate will develop its own unique set of mutations, making it difficult to identify a signature mutation that can effectively predict CCR5 antagonist resistance. This may explain why we do not observe shared mutations among clinical studies. The present study examined the earliest events in the development of drug resistance with viral quasispecies that continued the use of CCR5 for entry. Genotypic and phenotypic assays demonstrated a distinct role of the variable domain V1/V2 in competitive resistance to CCR5 antagonist therapy. Thus, future studies analyzing the development of clinical resistance should focus on the relationship between the V1/V2 and V3 domains.

  • HIV-1 Subtype C with PYxE Insertion Has Enhanced Binding of Gag-p6 to Host Cell Protein ALIX and Increased Replication Fitness [Pathogenesis and Immunity]

  • Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C-infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when the PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEi viruses and inserted them in a reference HIV-1 sequence. Viral growth was increased, and the sensitivity to the protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to that of wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion indicates the clinical importance of these viral variants.

    IMPORTANCE Genomic differences within HIV-1 subtypes is associated with various degrees of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1C has a natural deletion of one of these two motifs, resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of the antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

  • Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles [Vaccines and Antiviral Agents]

  • Most individuals are infected with respiratory syncytial virus (RSV) by age two, but infection does not result in long-term protective immunity to subsequent infections. Previous RSV infection may, however, impact responses to an RSV vaccine. The goal of these studies was to explore the effect of previous RSV infection on murine antibody responses to RSV F and G protein-containing virus-like particles (VLP), comparing responses to those resulting from VLP immunization of RSV-naive animals. These studies showed that after RSV infection, immunization with a single dose of VLPs containing a conformation-stabilized prefusion F protein stimulated high titers of neutralizing antibodies (NA), while an immunization with post-F-containing VLPs or a second RSV infection only weakly stimulated NA, even though total anti-F protein IgG antibody levels in both VLP-immunized animals were similar. Furthermore, single pre-F or post-F VLP immunization of animals previously infected (primed) with RSV resulted in total anti-F antibody titers that were 10- to 12-fold higher than titers after a VLP prime and boost of RSV-naive animals or after two consecutive RSV infections. The avidities of serum antibodies as well as numbers of splenic B cells and bone marrow cells after different immunization protocols were also assessed. The combined results show that RSV infection can quite effectively prime animals for the production of protective antibodies that can be efficiently activated by a pre-F VLP boost but not by a post-F VLP boost or a second RSV infection.

    IMPORTANCE Humans may experience repeated infections caused by the same serotype of respiratory syncytial virus (RSV), in contrast to infections with most other viruses, indicating that immune memory responses to RSV are defective. However, the effects of any residual but nonprotective immunity on responses to RSV vaccines are not clear. This study demonstrates that a VLP vaccine candidate containing a stabilized prefusion F protein can robustly stimulate protective immunity in animals previously infected with RSV, while a second RSV infection or a postfusion F-containing VLP cannot. This result shows that a properly constructed immunogen can be an effective vaccine in animals previously infected with RSV. The results also suggest that the defect in RSV memory is not in the induction of that memory but rather in its activation by a subsequent RSV infection.

  • Modeling Herpes Simplex Virus 1 Infections in Human Central Nervous System Neuronal Cells Using Two- and Three-Dimensional Cultures Derived from Induced Pluripotent Stem Cells [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcomes of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced pluripotent stem cells (hiPSCs) and neurons derived from them are documented as tools to study aspects of neuropathogenesis, but few have focused on modeling infections of the CNS. Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1nndash;humannndash;CNS interactions. Our results show that (i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; (ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; (iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; and (iv) the organoids support reactivation of HSV-1, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.

    IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina, providing a model to study HSV-1 trafficking through complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems; though, in contrast to 2D cultures, it appeared to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together, our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1nndash;CNS interactions in a human context.

  • Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22 [Structure and Assembly]

  • Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, 29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.

    IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

  • CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes [Virus-Cell Interactions]

  • The ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3 (ggt;10-fold) and HIV-1LAI (2- to 5-fold) in multiple human cell lines and primary CD4+ T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3 but not HIV-1LAI. The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAI selected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAI but not HIV-1NL4-3. The rescue of N57A by G94D in HIV-1LAI is abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3 and HIV-1LAI CA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.

    IMPORTANCE The specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

  • Selection and Characterization of a Reovirus Mutant with Increased Thermostability [Structure and Assembly]

  • The environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual-layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) 3 is degraded to generate the infectious subviral particle, and (ii) mmu;1 facilitates membrane penetration and subsequent core delivery. mmu;1-3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (mmu;1 M258I and 3 S344P) within heat-resistant strains. 3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability.

    IMPORTANCE Nonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, these findings show capsid function is balanced for optimal replication and for spread to a new host.

  • Articles of Significant Interest in This Issue [Spotlight]

  • Persistent Replication of HIV, Hepatitis C Virus (HCV), and HBV Results in Distinct Gene Expression Profiles by Human NK Cells [Cellular Response to Infection]

  • Natural killer (NK) cells during chronic viral infection have been well studied in the past. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV, and HBV viremia on NK cell transcriptomes. Using cell sorting, we obtained CD3nndash; CD56+ NK cells from blood of 6 HIV-, 8 HCV-, and 32 HBV-infected patients without treatment. After library preparation and sequencing, we used an in-house analytic pipeline to compare expression levels with matched healthy controls. In NK cells from HIV-, HCV-, and HBV-infected patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change, ggt;1.5; q-value, 0.2). Interferon-stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV-infected patients, viral load and alanine aminotransferase (ALT) variation had little effect on genes related to NK effector function. In conclusion, we compare, for the first time, NK cell transcripts of viremic HIV, HCV, and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in three different populations, suggestive for a different degree of functional alterations of the NK cell compartment compared to healthy individuals.

    IMPORTANCE Three viruses exist that can result in persistently high viral loads in immunocompetent humans: human immunodeficiency virus (HIV), hepatitis C virus, and hepatitis B virus. In the last decades, by using flow cytometry and in vitro assays on NK cells from patients with these types of infections, several impairments have been established, particularly during and possibly contributing to HIV viremia. However, the background of NK cell impairments in viremic patients is not well understood. In this study, we describe the NK cell transcriptomes of patients with high viral loads of different etiologies. We clearly demonstrate distinctive NK cell gene signatures with regard to interferon-stimulated gene induction and the expression of genes coding for activation markers or proteins involved in cytotoxic action, as well immunological genes. This study provides important details necessary to uncover the origin of functional and phenotypical differences between viremic patients and healthy subjects and provides many leads that can be confirmed using future in vitro manipulation experiments.

  • DNA Tumor Viruses and Their Contributions to Molecular Biology [Gem]

  • This summer marks the 51st anniversary of the DNA tumor virus meetings. Scientists from around the world will gather in Trieste, Italy, to report their latest results and to agree or disagree on the current concepts that define our understanding of this diverse class of viruses. This article offers a brief history of the impact the study of these viruses has had on molecular and cancer biology and discusses obstacles and opportunities for future progress.

  • Structure of Parvovirus B19 Decorated by Fabs from a Human Antibody [Structure and Assembly]

  • Parvovirus B19, one of the most common human pathogens, is a small DNA virus that belongs to the Parvoviridae. As a result of previous infections, antibodies to B19 are present in most adults. B19 has a strong tropism to erythroid progenitor cells and is able to cause a series of medical conditions, including fifth disease, arthritis, myocarditis, hydrops fetalis, and aplastic crisis. No approved vaccine is currently available for B19, and there is a lack of structural characterization of any B19 epitopes. Here we present the first cryo-electron microscopy (cryo-EM) structure of a B19 virus-like particle (VLP) complexed with the antigen-binding fragment (Fab) of a human neutralizing antibody, 860-55D. A model was built into the 3.2-AAring;-resolution map, and the antigenic residues on the surface of the B19 capsid were identified. Antibody 860-55D bridges the capsid of B19 by binding to a quaternary structure epitope formed by residues from three neighboring VP2 capsid proteins.

    IMPORTANCE Parvovirus B19 is a common human pathogen and a particular threat to children, pregnant women, and patients with sickle cell disease or AIDS. Currently, neutralizing antibody is the most efficient treatment for acute B19 infections. Research on the antigenic properties of B19 will guide the usage of these antibodies and facilitate vaccine development. We have determined and report here the high-resolution structure of B19 virus-like particles (VLPs) complexed with the Fab of a human neutralizing antibody. The structure shows a quaternary structure epitope formed by three VP2 proteins and provides details on host recognition of human B19 virus.

  • Manipulation of Aphid Behavior by a Persistent Plant Virus [Cellular Response to Infection]

  • Plants are frequently infected with cytoplasmic RNA viruses that persist for many generations through nearly 100% vertical transmission without producing any symptoms. Movement between plant cells and horizontal transmission have not been observed with these viruses; instead, they are distributed to all host cells through host cell division. Jalapenntilde;o peppers (Capsicum annuum) are all infected with Pepper cryptic virus 1 (PCV-1; family Partitiviridae). We compared the effect of odor cues from PCV-1-infected (J+) and virus-free (Jnndash;) jalapenntilde;o peppers on the aphid Myzus persicae, a common vector of acute plant viruses. Pairwise preference experiments showed a stark contrast to insect-plant interactions in acute virus infectionsmmdash;that is, the virus-infected plants deterred aphids. The acute plant virus Cucumber mosaic virus (CMV) manipulates its host's volatile emissions to attract aphid vectors and facilitate its transmission. We inoculated J+ and Jnndash; plants with CMV. Volatiles of J+ and Jnndash; CMV-infected plants were more attractive to aphids than those of J+ and Jnndash; mock-inoculated plants. However, in pairwise preference experiments with J+ CMV- and Jnndash; CMV-infected plants, aphids preferred the Jnndash; CMV volatile blend. Aphid reproduction on J+ and Jnndash; plants was measured as an indicator of the effect of PCV-1 on host quality for aphids. Aphid reproduction on J+ plants was more than 2-fold lower than that on Jnndash; plants.

    IMPORTANCE This study demonstrates that a persistent plant virus can manipulate aphid behavior. This manipulation is in stark contrast to previously described effects of acute viruses on their hosts that facilitate their transmission. This study demonstrates a positive relationship between Pepper cryptic virus 1 and jalapenntilde;o pepper (Capsicum annuum) plants wherein the virus protects the plants from the vector of acute viruses and reduces aphid herbivory. This work reveals an important implication of persistent plant viruses for pest and pathogen management in agriculture.

  • HACE1, an E3 Ubiquitin Protein Ligase, Mitigates Kaposis Sarcoma-Associated Herpesvirus Infection-Induced Oxidative Stress by Promoting Nrf2 Activity [Virus-Cell Interactions]

  • Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV)-induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is essential for both the expression of viral genes (latency) and modulation of the host antioxidant machinery. Reactive oxygen species (ROS) are also regulated by the ubiquitously expressed HACE1 protein (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1), which targets the Rac1 protein for proteasomal degradation, and this blocks the generation of ROS by Rac1-dependent NADPH oxidases. In this study, we examined the role of HACE1 in KSHV infection. Elevated levels of HACE1 expression were observed in de novo KSHV-infected endothelial cells, KSHV latently infected TIVE-LTC and PEL cells, and Kaposirrsquo;s sarcoma skin lesion cells. The increased HACE1 expression in the infected cells was mediated by KSHV latent protein kaposin A. HACE1 knockdown resulted in high Rac1 and Nox 1 (NADPH oxidase 1) activity, increased ROS (oxidative stress), increased cell death, and decreased KSHV gene expression. Loss of HACE1 impaired KSHV infection-induced phosphoinositide 3-kinase (PI3-K), protein kinase C- (PKC-), extracellular signal-regulated kinase 1/2 (ERK1/2), NF-B, and Nrf2 activation and nuclear translocation of Nrf2, and it reduced the expression of Nrf2 target genes responsible for balancing the oxidative stress. In the absence of HACE1, glutamine uptake increased in the cells to cope with the KSHV-induced oxidative stress. These findings reveal for the first time that HACE1 plays roles during viral infection-induced oxidative stress and demonstrate that HACE1 facilitates resistance to KSHV infection-induced oxidative stress by promoting Nrf2 activity. Our studies suggest that HACE1 could be a potential target to induce cell death in KSHV-infected cells and to manage KSHV infections.

    IMPORTANCE ROS play important roles in several cellular processes, and increased ROS cause several adverse effects. KSHV infection of endothelial cells induces ROS, which facilitate virus entry by amplifying the infection-induced host cell signaling cascade, which, in turn, induces the nuclear translocation of phospho-Nrf2 protein to regulate the expression of antioxidative genes and viral genes. The present study demonstrates that KSHV infection induces the E3 ligase HACE1 protein to regulate KSHV-induced oxidative stress by promoting the activation of Nrf2 and nuclear translocation. Absence of HACE1 results in increased ROS and cellular death and reduced nuclear Nrf2, antioxidant, and viral gene expression. Together, these studies suggest that HACE1 can be a potential target to induce cell death in KSHV-infected cells.

  • Turnip Mosaic Virus Is a Second Example of a Virus Using Transmission Activation for Plant-to-Plant Propagation by Aphids [Virus-Cell Interactions]

  • Cauliflower mosaic virus (CaMV; family Caulimoviridae) responds to the presence of aphid vectors on infected plants by forming specific transmission morphs. This phenomenon, coined transmission activation (TA), controls plant-to-plant propagation of CaMV. A fundamental question is whether other viruses rely on TA. Here, we demonstrate that transmission of the unrelated turnip mosaic virus (TuMV; family Potyviridae) is activated by the reactive oxygen species H2O2 and inhibited by the calcium channel blocker LaCl3. H2O2-triggered TA manifested itself by the induction of intermolecular cysteine bonds between viral helper component protease (HC-Pro) molecules and by the formation of viral transmission complexes, composed of TuMV particles and HC-Pro that mediates vector binding. Consistently, LaCl3 inhibited intermolecular HC-Pro cysteine bonds and HC-Pro interaction with viral particles. These results show that TuMV is a second virus using TA for transmission but using an entirely different mechanism than CaMV. We propose that TuMV TA requires reactive oxygen species (ROS) and calcium signaling and that it is operated by a redox switch.

    IMPORTANCE Transmission activation, i.e., a viral response to the presence of vectors on infected hosts that regulates virus acquisition and thus transmission, is an only recently described phenomenon. It implies that viruses contribute actively to their transmission, something that has been shown before for many other pathogens but not for viruses. However, transmission activation has been described so far for only one virus, and it was unknown whether other viruses also rely on transmission activation. Here we present evidence that a second virus uses transmission activation, suggesting that it is a general transmission strategy.

  • The I{kappa}B Kinases Restrict Human Cytomegalovirus Infection [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes disease in immunosuppressed populations. HCMV has a complex relationship with innate immune signaling pathways. Specifically, HCMV has been found to block some aspects of inflammatory signaling while benefiting from others. Through analysis of knockout cell lines targeting the NF-B regulatory kinases IB kinase aalpha; (IKKaalpha;) and IKKbbeta;, we find that the IKKs are host restriction factors that contribute to cytokine-mediated resistance to viral infection, limit the initiation of HCMV infection, and attenuate viral cell-to-cell spread. The HCMV UL26 protein is a viral immune modulator important for HCMV infection that has been shown to inhibit host cell NF-B signaling, yet it has remained unclear how UL26-mediated NF-B modulation contributes to infection. Here, we find that UL26 modulation of NF-B signaling is separable from its contribution to high-titer viral replication. However, we find that IKKbbeta; is required for the induction of cytokine expression associated with UL26 infection. Collectively, our data indicate that the IKKs restrict infection but HCMV targets their signaling to modulate the cellular inflammatory environment.

    IMPORTANCE Innate immune signaling is a critical defense against viral infection and represents a central host-virus interaction that frequently determines the outcomes of infections. NF-B signaling is an essential component of innate immunity that is extensively modulated by HCMV, a significant cause of morbidity in neonates and immunosuppressed individuals. However, the roles that various facets of NF-B signaling play during HCMV infection have remained elusive. We find that the two major regulatory kinases in this pathway, IKKaalpha; and IKKbbeta;, limit the initiation of infection, viral replication, and cell-to-cell spread. In addition, our results indicate that these kinases contribute differently to the host cell response to infection in the absence of a virally encoded NF-B inhibitor, UL26. Given the importance of NF-B in viral infection, elucidating the contributions of various NF-B constituents to infection is an essential first step toward the possibility of targeting this pathway therapeutically.

  • Mapping the Heterogeneity of Histone Modifications on Hepatitis B Virus DNA Using Liver Needle Biopsies Obtained from Chronically Infected Patients [Genome Replication and Regulation of Viral Gene Expression]

  • Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. We have previously shown that viral transcription is subject to regulation by posttranslational modifications (PTMs) of histone proteins bound to cccDNA through analysis of de novo HBV-infected cell lines. We now report the successful adaptation of this chromatin immunoprecipitation sequencing (ChIPseq) approach for analysis of fine-needle patient liver biopsy specimens to investigate the role of histone PTMs in chronically HBV-infected patients. Using 18 specimens from patients in different stages of chronic HBV infection, our work shows that the profile of histone PTMs in chronic infection is more nuanced than previously observed in in vitro models of acute infection. In line with our previous findings, we find that the majority of HBV-derived sequences are associated with the activating histone PTM H3K4me3. However, we show a striking interpatient variability of its deposition in this patient cohort correlated with viral transcription and patient HBV early antigen (HBeAg) status. Unexpectedly, we detected deposition of the classical inhibitory histone PTM H3K9me3 on HBV-DNA in around half of the patient biopsy specimens, which could not be linked to reduced levels of viral transcripts. Our results show that current in vitro models are unable to fully recapitulate the complex epigenetic landscape of chronic HBV infection observed in vivo and demonstrate that fine-needle liver biopsy specimens can provide sufficient material to further investigate the interaction of viral and host proteins on HBV-DNA.

    IMPORTANCE Hepatitis B virus (HBV) is a major global health concern, chronically infecting millions of patients and contributing to a rising burden of liver disease. The viral genome forms the basis for chronic infection and has been shown to be subject to regulation by epigenetic mechanisms, such as posttranslational modification of histone proteins. Here, we confirm and expand on previous results by adapting a high-resolution technique for analysis of histone modifications for use with patient-derived fine-needle liver biopsy specimens. Our work highlights that the situation in vivo is more complex than predicted by current in vitro models, for example, by suggesting a novel, noncanonical role of the histone modification H3K9me3 in the HBV life cycle. Importantly, enabling the use of fine-needle liver biopsy specimens for such high-resolution analyses may facilitate further research into the epigenetic regulation of the HBV genome.

  • The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity [Virus-Cell Interactions]

  • The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin aalpha; isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus.

    IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin aalpha; protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin aalpha; binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

  • Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5' Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA [Structure and Assembly]

  • Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227nndash;2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255nndash;12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that ggt;90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only llt;1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.

    IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227nndash;2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255nndash;12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

  • A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention [Virus-Cell Interactions]

  • Chromatin-based modifications of herpesviral genomes play a crucial role in dictating the outcome of infection. Consistent with this, host cell multiprotein complexes, such as polycomb repressive complexes (PRCs), were proposed to act as epigenetic regulators of herpesviral latency. In particular, PRC2 has recently been shown to contribute to the silencing of human cytomegalovirus (HCMV) genomes. Here, we identify a novel proviral role of PRC1 and PRC2, the two main polycomb repressive complexes, during productive HCMV infection. Western blot analyses revealed strong HCMV-mediated upregulation of RING finger protein 1B (RING1B) and B lymphoma Moloney murine leukemia virus insertion region 1 homolog (BMI1) as well as of enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED), which constitute the core components of PRC1 and PRC2, respectively. Furthermore, we observed a relocalization of PRC components to viral replication compartments, whereas histone modifications conferred by the respective PRCs were specifically excluded from these sites. Depletion of individual PRC1/PRC2 proteins by RNA interference resulted in a significant reduction of newly synthesized viral genomes and, in consequence, a decreased release of viral particles. Furthermore, accelerated native isolation of protein on nascent DNA (aniPOND) revealed a physical association of EZH2 and BMI1 with nascent HCMV DNA, suggesting a direct contribution of PRC proteins to viral DNA replication. Strikingly, substances solely inhibiting the enzymatic activity of PRC1/2 did not exert antiviral effects, while drugs affecting the abundance of PRC core components strongly compromised HCMV genome synthesis and particle release. Taken together, our data reveal an enzymatically independent, noncanonical function of both PRC1 and PRC2 during HCMV DNA replication, which may serve as a novel cellular target for antiviral therapy.

    IMPORTANCE Polycomb group (PcG) proteins are primarily known as transcriptional repressors that modify chromatin and contribute to the establishment and maintenance of cell fates. Furthermore, emerging evidence indicates that overexpression of PcG proteins in various types of cancers contributes to the dysregulation of cellular proliferation. Consequently, several inhibitors targeting PcG proteins are presently undergoing preclinical and clinical evaluation. Here, we show that infection with human cytomegalovirus also induces a strong upregulation of several PcG proteins. Our data suggest that viral DNA replication depends on a noncanonical function of polycomb repressor complexes which is independent of the so-far-described enzymatic activities of individual PcG factors. Importantly, we observe that a subclass of inhibitory drugs that affect the abundance of PcG proteins strongly interferes with viral replication. This principle may serve as a novel promising target for antiviral treatment.

  • Simian Immunodeficiency Virus Infects Functionally Polarized Memory CD4 T Cells Equivalently In Vivo [Pathogenesis and Immunity]

  • Among the numerous immunological abnormalities observed in chronically human immunodeficiency virus (HIV)-infected individuals, perturbations in memory CD4 T cells are thought to contribute specifically to disease pathogenesis. Among these, functional imbalances in the frequencies of T regulatory cells (Tregs) and interleukin 17 (IL-17)/IL-22-producing Th cells (Th17/Th22) from mucosal sites and T follicular helper (Tfh) cells in lymph nodes are thought to facilitate specific aspects of disease pathogenesis. However, while preferential infection of Tfh cells is widely thought to create an important viral reservoir in an immunologically privileged site in vivo, whether immunological perturbations among memory CD4 T cell populations are attributable to their relative infectivity by the virus in vivo is unclear. Here we studied peripheral blood and lymphoid tissues from antiretroviral (ARV)-treated and ARV-naive Asian macaques and isolated functionally defined populations of memory CD4 T cells. We then assessed the degree to which these populations were infected by simian immunodeficiency virus (SIV) in vivo, to determine whether particular functionally identified populations of memory CD4 T cells were preferentially infected by the virus. We found that SIV did not preferentially infect Th17 cells, compared to Th1 cells, Th2 cells, or Tregs. Moreover, Th17 cells contributed proportionately to the total pool of infected cells. Taken together, our data suggest that, although Tfh cells are more prone to harbor viral DNA, other functionally polarized cells are equally infected by the virus in vivo and Th17 cells are not preferentially infected.

    IMPORTANCE Functional perturbations of memory CD4 T cells have been suggested to underlie important aspects of HIV disease progression. However, the mechanisms underlying these perturbations remain unclear. Using a nonhuman primate model of HIV, we show that SIV infects functionally defined populations of memory CD4 T cells equally in different anatomic sites. Thus, preferential infection by the virus is unlikely to cause functional perturbations.

  • The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection [Pathogenesis and Immunity]

  • Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the aalpha; subunit of eukaryotic initiation factor 2 (eIF2aalpha;). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo. In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2aalpha; phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2aalpha; unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2aalpha; correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.

    IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo. This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

  • Adaptation of an R5 Simian-Human Immunodeficiency Virus Encoding an HIV Clade A Envelope with or without Ablation of Adaptive Host Immunity: Differential Selection of Viral Mutants [Pathogenesis and Immunity]

  • Simian-human immunodeficiency virus (SHIV) infection in rhesus macaques (RMs) resembles human immunodeficiency virus type 1 (HIV-1) infection in humans and serves as a tool to evaluate candidate AIDS vaccines. HIV-1 clade A (HIV-A) predominates in parts of Africa. We constructed an R5 clade A SHIV (SHIV-A; strain SHIV-KNH1144) carrying env from a Kenyan HIV-A. SHIV-A underwent rapid serial passage through six RMs. To allow unbridled replication without adaptive immunity, we simultaneously ablated CD8+ and B cells with cytotoxic monoclonal antibodies in the next RM, resulting in extremely high viremia and CD4+ T-cell loss. Infected blood was then transferred into two non-immune-depleted RMs, where progeny SHIV-A showed increased replicative capacity and caused AIDS. We reisolated SHIV-KNH1144p4, which was replication competent in peripheral blood mononuclear cells (PBMC) of all RMs tested. Next-generation sequencing of early- and late-passage SHIV-A strains identified mutations that arose due to "fitness" virus optimization in the former and mutations exhibiting signatures typical for adaptive host immunity in the latter. "Fitness" mutations are best described as mutations that allow for better fit of the HIV-A Env with SIV-derived virion building blocks or host proteins and mutations in noncoding regions that accelerate virus replication, all of which result in the outgrowth of virus variants in the absence of adaptive T-cell and antibody-mediated host immunity.

    IMPORTANCE In this study, we constructed a simian-human immunodeficiency virus carrying an R5 Kenyan HIV-1 clade A env (SHIV-A). To bypass host immunity, SHIV-A was rapidly passaged in naive macaques or animals depleted of both CD8+ and B cells. Next-generation sequencing identified different mutations that resulted from optimization of viral replicative fitness either in the absence of adaptive immunity or due to pressure from adaptive immune responses.

  • Genome Characterization, Prevalence, and Transmission Mode of a Novel Picornavirus Associated with the Threespine Stickleback Fish (Gasterosteus aculeatus) [Structure and Assembly]

  • The complete genome sequence of an RNA virus was assembled from RNA sequencing of virus particles purified from threespine stickleback intestine tissue samples. This new virus is most closely related to the Eel picornavirus and can be assigned to the genus Potamipivirus in the family Picornaviridae. Its unique genetic properties are enough to establish a new species, dubbed the Threespine Stickleback picornavirus (TSPV). Due to their broad geographic distribution throughout the Northern Hemisphere and parallel adaptation to freshwater, threespine sticklebacks have become a model in evolutionary ecology. Further analysis using diagnostic PCRs revealed that TSPV is highly prevalent in both anadromous and freshwater populations of threespine sticklebacks, infects almost all fish tissues, and is transmitted vertically to offspring obtained from in vitro fertilization in laboratory settings. Finally, TSPV was found in Sequence Reads Archives of transcriptome of Gasterosteus aculeatus, further demonstrating its wide distribution and unsought prevalence in samples. It is thus necessary to test the impact of TSPV on the biology of threespine sticklebacks, as this widespread virus could interfere with the behavioral, physiological, or immunological studies that employ this fish as a model system.

    IMPORTANCE The threespine stickleback species complex is an important model system in ecological and evolutionary studies because of the large number of isolated divergent populations that are experimentally tractable. For similar reasons, its coevolution with the cestode parasite Schistocephalus solidus, its interaction with gut microbes, and the evolution of its immune system are of growing interest. Herein we describe the discovery of an RNA virus that infects both freshwater and anadromous populations of sticklebacks. We show that the virus is transmitted vertically in laboratory settings and found it in Sequence Reads Archives, suggesting that experiments using sticklebacks were conducted in the presence of the virus. This discovery can serve as a reminder that the presence of viruses in wild-caught animals is possible, even when animals appear healthy. Regarding threespine sticklebacks, the impact of Threespine Stickleback picornavirus (TSPV) on the fish biology should be investigated further to ensure that it does not interfere with experimental results.

  • Comparison of the Whole-Genome Sequence of an Oka Varicella Vaccine from China with Other Oka Vaccine Strains Reveals Sites Putatively Critical for Vaccine Efficacy [Vaccines and Antiviral Agents]

  • Varicella-zoster virus (VZV) infection results in varicella mostly in children. Reactivation of the virus causes herpes zoster (HZ), mostly in adults. A live attenuated vaccine (vOka-Biken) was originally derived from the parental strain pOka. Several live attenuated vaccines based on the Oka strain are currently available worldwide. In China, varicella vaccines have been licensed by four manufacturers. In this study, we analyze the whole-genome sequence (WGS) of vOka-BK produced by Changchun BCHT Biotechnology also known as Baike. vOka-BK WGS was compared against the genomic sequences of four other Oka strains: pOka, vOka-Biken, vOka-Varilrix from GlaxoSmithKline, and vOka-Varivax from Merck aamp; Co. A previous study identified 137 single nucleotide polymorphisms (SNPs) shared by all vOkas. The current analysis used these data as a reference to compare with vOka-BK WGS and focused on 54 SNPs located in the unique regions of the genome. Twenty-eight nonsynonymous substitutions were identified, ORF62 and ORF55 featuring the most amino acid changes with 9 and 3, respectively. Among the 54 SNPs, 10 had a different mutation profile in vOka-BK compared to the other three vaccines. A comparison with the clade 3 strain Ellen, known to be attenuated, identified three shared amino acid changes: *130R in ORF0 and R958G and S628G in ORF62. This analysis provides the first comparison of a Chinese varicella vaccine to the other vaccines available worldwide and identifies sites potentially critical for VZV vaccine efficacy.

    IMPORTANCE Varicella, also known as chickenpox, is a highly contagious disease, caused by varicella-zoster virus (VZV). Varicella is a common childhood disease that can be prevented by a live attenuated vaccine. The first available vaccine was derived from the parental Oka strain in Japan in 1974. Several live attenuated vaccines based on the Oka strain are currently available worldwide. Among the four vaccines produced in China, the vaccine manufactured by Changchun BCHT Biotechnology, also known as Baike, has been reported to be very efficacious. Comparative genomic analysis of the Baike vaccine with other Oka vaccine strains identified sites that might be involved in vaccine efficacy, as well as important for the biology of the virus.

  • Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3 [Virus-Cell Interactions]

  • Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marekrrsquo;s disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication.

    IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

  • Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes [Vaccines and Antiviral Agents]

  • Reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The immune mechanisms of protection against recurrent genital herpes remain to be fully elucidated. In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed HSV-2 envelope and tegument proteins. These viral protein antigens (Ags) were rationally selected for their ability to recall strong CD4+ and CD8+ T-cell responses from naturally "protected" asymptomatic individuals, who, despite being infected, never develop any recurrent herpetic disease. Out of the eight HSV-2 proteins, the envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene), and ribonucleotide reductase subunit 2 protein (RR2; encoded by the UL40 gene) produced significant protection against recurrent genital herpes. The RR2 protein, delivered either intramuscularly or intravaginally with CpG and alum adjuvants, (i) boosted the highest neutralizing antibodies, which appear to cross-react with both gB and gD, and (ii) enhanced the numbers of functional gamma interferon (IFN-)-producing CRTAM+ CFSE+ CD4+ and CRTAM+ CFSE+ CD8+ TRM cells, which express low levels of PD-1 and TIM-3 exhaustion markers and were localized to healed sites of the vaginal mucocutaneous (VM) tissues. The strong B- and T-cell immunogenicity of the RR2 protein was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In vivo depletion of either CD4+ or CD8+ T cells significantly abrogated the protection. Taken together, these preclinical results provide new insights into the immune mechanisms of protection against recurrent genital herpes and promote the tegument RR2 protein as a viable candidate Ag to be incorporated in future genital herpes therapeutic mucosal vaccines.

    IMPORTANCE Recurrent genital herpes is one of the most common sexually transmitted diseases, with a global prevalence of HSV-2 infection predicted to be over 536 million worldwide. Despite the availability of many intervention strategies, such as sexual behavior education, barrier methods, and the costly antiviral drug treatments, eliminating or at least reducing recurrent genital herpes remains a challenge. Currently, no FDA-approved therapeutic vaccines are available. In this preclinical study, we investigated the immunogenicity and protective efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed herpes envelope and tegument proteins. We discovered that similar to the dl5-29 vaccine, based on a replication-defective HSV-2 mutant virus, which has been recently tested in clinical trials, the RR2 protein-based subunit vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with an increase in numbers of functional tissue-resident IFN-+ CRTAM+ CFSE+ CD4+ and IFN-+ CRTAM+ CFSE+ CD8+ TRM cells that infiltrate healed sites of the vaginal tissues. Our study sheds new light on the role of TRM cells in protection against recurrent genital herpes and promotes the RR2-based subunit therapeutic vaccine to be tested in the clinic.

  • Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation [Virus-Cell Interactions]

  • Enterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IRES trans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.

    IMPORTANCE Outbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

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  • Phenotypic and functional differences between HHV-6 and HCMV specific T-cells [Pathogenesis and Immunity]

  • Background: Human herpesvirus 6 (HHV-6) infects over 90% of the population, and establishes a latent infection, with asymptomatic episodes of reactivation. However, HHV-6 reactivation is associated with morbidity and sometimes mortality in immunocompromised patients. To date, the control of the virus in healthy virus carriers and its failure in patients with disease remain poorly understood. In particular, knowledge on HHV-6 specific T-cell responses is limited.

    Methods: Here, we characterized HHV-6A and 6B specific CD4+ and CD8+ T-cell responses from peripheral blood mononuclear cells (PBMCs) of healthy donors. We studied the phenotype of effector HHV-6 specific T-cells ex vivo, as well as of induced specific suppressive regulatory CD4+ T-cells in vitro post-stimulation, in comparison to human cytomegalovirus (HCMV) responses.

    Results: Compared to HCMV, we show that ex vivo T-cell reactivity in peripheral blood is detectable but at very low frequency, both for HHV-6A and 6B viruses. Interestingly, the phenotype of the specific T-cells is also different between both viruses. HHV-6A and 6B specific CD4+ T lymphocytes are less differentiated than HCMV specific T-cells. Furthermore, we show a higher frequency of HHV-6 specific suppressive Treg (eTreg) than HCMV specific eTreg in co-infected individuals.

    Conclusions: Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, in particular related to the frequency and phenotype of effector/memory and regulatory virus specific T-cells. This suggests that different immune actors are solicited in the control of HHV-6 compared to HCMV infection.

    Importance

    T-cells are central to an effective defense against persistent viral infections that can be related to human cytomegalovirus (HCMV) or human herpes virus 6 (HHV-6). However, knowledge on HHV-6 specific T-cell responses is limited.

    In order to deepen our knowledge on T-cell responses to HHV-6, we have characterized the HHV-6A and 6B specific CD4+ and CD8+ T-cell responses directly ex vivo from healthy co-infected blood donors.

    Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, in particular related to the frequency and phenotype of effector/memory and regulatory virus specific T-cells. This suggests that different immune actors are solicited in the control of HHV-6 compared to HCMV infection.

    Our findings may encourage immunomonitoring of patients with viral replication episodes to follow the emergence of effector vs regulatory T cells.

  • Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next Generation Vaccine Candidate [Vaccines and Antiviral Agents]

  • Enterovirus A71 (EV-A71) is the major pathogen which causes hand-foot-and-mouth disease (HFMD) which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon deoptimized VP1 capsid protein (VP1-CD) showed similar replication rate and antigenicity to wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques, but was still able to examined by TCID50 titers which were similar to the others, thus indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF) and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in mice infection model. After 14 days post-immunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates proof-of-concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, which may be a good candidate for next generation EV-A71 vaccine development.

    IMPORTANCE Enterovirus A71 (EV-A71) can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here we developed a novel vaccine strategy with the combination of codon-deoptimization (CD) and high-fidelity (HF) substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated that the simultaneous introduction of CD genome sequences and HF substitutions as a potentially new strategy to develop attenuated vaccine seed virus. Our work provided an insight to develop the low virulent candidate vaccine virus (CVV) through a series of genetic editing on virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next generation vaccine development of EV-A71.

  • Regulation of human cytomegalovirus secondary envelopment by a C-terminal tetra-lysine motif in pUL71 [Structure and Assembly]

  • Human cytomegalovirus (HCMV) secondary envelopment requires the viral tegument protein pUL71. Lack of pUL71 results in a complex ultrastructural phenotype with increased numbers of viral capsids undergoing envelopment at the cytoplasmic virus assembly complex. Here, we report a role of the pUL71 C-terminus in secondary envelopment. Mutant viruses expressing C-terminally truncated pUL71 (TBdel327-361 and TB71del348-351) exhibited an impaired secondary envelopment in transmission electron microscopy (TEM) studies. Further mutational analyses of the C-terminus revealed a tetra-lysine motif whose mutation (TB71mutK348-351A) resulted in an envelopment defect that was undistinguishable from the defect caused by truncation of the pUL71 C-terminus. Interestingly, not all morphological alterations that define the ultrastructural phenotype of a TB71stop virus were found in cells infected with the C-terminally mutated viruses. This suggests that pUL71 provides additional functions that modulate HCMV morphogenesis and are encoded elsewhere in pUL71. This is also reflected by an intermediate growth defect of the C-terminally mutated viruses compared to the TB71stop virus. Electron tomography and three-dimensional visualization of different stages of secondary envelopment in TB71mutK348-351A infected cells showed unambiguously the formation of a bud neck. Furthermore, we provide evidence for a progressive tegument formation linked to advancing grades of capsid envelopment, suggesting that tegumentation and envelopment are intertwined processes. Together, we identified the importance of the pUL71 C-terminus and specifically of a positively-charged tetra-lysine motif for HCMV secondary envelopment.

    IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes severe symptoms especially in immunocompromised hosts. Furthermore, congenital HCMV infection is the leading viral cause of severe birth defects. Development of antiviral drugs to prevent production of infectious virus progeny is challenging due to a complex and multi-step virion morphogenesis. The mechanism of secondary envelopment is still not fully understood, nevertheless, it represents a potential target for antiviral drugs. Our identification of the role of a positively charged motif in the pUL71 C-terminus for efficient HCMV secondary envelopment underlines the importance of pUL71 and especially its C-terminus for this process. It furthermore shows how cell-associated spread and virion release depends on secondary envelopment. Ultrastructural analyses of different stages of envelopment contribute to a better understanding of the mechanisms underlying the process of secondary envelopment. This may bring us closer to develop novel concepts to treat HCMV infections.

  • Carnivore parvovirus ecology in the Serengeti ecosystem: vaccine strains circulating and new host species identified [Genetic Diversity and Evolution]

  • Carnivore parvoviruses infect wild and domestic carnivores and cross- species transmission is believed to occur. However, viral dynamics are not well understood nor the consequences to wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multi-host system such as the Serengeti ecosystem and identify potential threats for wildlife conservation we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separated routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP); and second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized, new host-virus associations involving wild dogs, jackals and hyaenas were discovered and our results suggest mutations in the fragment of the vp2 gene were not required to infect different carnivore species. In domestic dogs, six sequences belonged to the CPV-2a strain, whilst 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.

    Importance of this study Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. The findings from this study, which further the understanding of carnivore parvovirus epidemiology, suggest that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP); further, that canine parvoviruses are present in the dog population living around the SNP, with little evidence of transmission into wild carnivore species; and finally, that the detection of vaccine-derived virus (described here for the first time circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.

  • A genome-wide haploid genetic screen identifies heparan sulfate-associated genes and the macropinocytosis modulator TMED10 as factors supporting vaccinia virus infection [Virus-Cell Interactions]

  • Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2 and TMED10 (TMP21/p23/p24) as important host factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.

    Importance

    Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells, and are used as vaccine vector against tuberculosis, influenza, and coronaviruses.

    Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knock-out library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

  • Asparagine is a critical limiting metabolite for vaccinia virus protein synthesis during glutamine deprivation [Virus-Cell Interactions]

  • Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Further, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of anti-viral strategy development.

    IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of poxviruses that comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine that is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene poxvirus infection.

  • Comparisons of human immunodeficiency virus type 1 envelope variants in blood and genital fluids near the time of male-to-female transmission [Pathogenesis and Immunity]

  • To better understand the transmission of human immunodeficiency virus type-1 (HIV-1), the genetic characteristics of male blood and genital viruses were compared to the imputed founding virus population in their female partners. Initially serodiscordant heterosexual African couples with sequence-confirmed male-to-female HIV-1 transmission and blood and genitial specimens near the time of transmission were studied. Single viral templates from blood plasma and genital tract RNA and DNA were sequenced across HIV-1 env gp160. Eight of twenty-nine couples examined yielded viral sequences from both tissues. Analysis of these couples' sequences demonstrated, with one exception, that the women's founding viral populations arose from a single viral variant, and were CCR5-tropic despite X4-variants detected within four males. The median genetic distance of the imputed MRCA of the women's founder viruses was closer to the semen than to the blood viruses of their transmitting male partner, but this finding was biased by detection of a greater number of viral clades in the blood. Using multiple assays, the males' blood and genital viruses were consistently compartmentalized in only two of eight men. No distinct amino acid signatures in the men's viruses were found to link to the women's founders, nor did the women's env have shorter variable loops or fewer N-linked glycosylation sites. The lack of selective factors, except for co-receptor tropism, is consistent with others' findings in male-to-female and high-risk transmissions. The infrequent compartmentization between transmitters' blood and semen viruses suggest that cell-free blood virus likely includes HIV-1 sequences representative of semen.

    Importance Mucosal transmissions account for the majority of HIV-1 infections. Identification of the viral characteristics associated with transmission would facilitate vaccine design. This study of HIV strains from transmitting males and their seroconverting female partners found that males' genital tract viruses were rarely distinct from blood variants. The imputed founder viruses in women were genetically similar to both blood and genital tract variants of their male partners, indicating a lack of evidence for genital tract specific lineages. These findings suggest that targeting vaccine responses to variants found in blood are likely to also protect from genital tract variants.

  • Discovery of small molecule inhibitors targeting the E3 ubiquitin ligase activity of the HSV-1 ICP0 protein using an in vitro high throughput screening assay. [Virus-Cell Interactions]

  • Herpes simplex virus-1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors but emergence of drug resistant mutants is observed frequently, highlighting the need for novel anti-viral therapies. Infected Cell Protein 0 (ICP0) of HSV-1 is encoded by an immediate-early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces degradation of targeted proteins. A ICP0 virus or mutants with deficiencies in the E3 ligase activity cannot counteract IFN-bbeta; induced restriction of viral infection, are highly immunogenic, avirulent and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high throughput screening assay, based on the auto-ubiquitination properties of ICP0, to identify small molecule inhibitors of the ICP0 E3 ubiquitin ligase activity. Through a pilot screening we identified nine compounds that displayed a dose-dependent inhibitory effect on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 using a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.

    IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased so has the number of immunocompromised individuals, who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to virus-acquired drug resistance. Thus, novel treatments need to be explored. Herein, we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because: (i) ICP0 is expressed before virus replication; (ii) it is essential for the infection in vivo; (iii) it is required for efficient reactivation of the virus from latency; (iv) inhibition of its E3 ligase activity will sustain host immune responses; and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

  • Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal degradation of VP1 and Attenuating its Antagonistic Role on the IFN-{beta} signaling pathway [Virus-Cell Interactions]

  • DnaJ Heat Shock Protein Family (Hsp40) Member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. The J domain of DNAJA3 [amino acids (aa) 1nndash;168] and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss-of-function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the IFN-bbeta; signaling pathway by inhibiting the phosphorylation, dimerization and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on IFN-bbeta; signaling pathway. Poly (I: C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared with that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-bbeta; signaling pathway.

    Importance

    This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study was also the first to clarify the mechanism by which VP1 suppressed IFN-bbeta; signaling pathway by inhibiting the phosphorylation, dimerization and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-bbeta; signaling pathway. Those data suggested that DNAJA3 played an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-bbeta; signaling pathway.

  • Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination [Genetic Diversity and Evolution]

  • Senecavirus A (SVA) is a re-emerging virus and recent evidence has emphasized the importance of SVA recombination in vivo on virus evolution. In this study, we report the development of an infectious cDNA clone for the SVA/HLJ/CHA/2016 strain. We have used this strain to develop a reporter virus expressing eGFP, which we then used to screen for a recombination-deficient SVA by an eGFP-retention assay. Sequencing of the virus that retained the eGFP following passage allowed us to identify the non-synonymous mutations (S460L alone and I212V/S460L in combination) in the RNA-dependent RNA polymerase (RdRp) region of the genome. We developed a Senecavirus specific cell culture based recombination assay, which we used to elucidate the role of RdRp in SVA recombination. Our results demonstrate that these two polymerase variants (S460L and I212/S460L) have reduced recombination capacity. These results indicate that the RdRp plays a central role in SVA replicative recombination. Notably, our results showed that the two recombination-deficient variants are of higher replication fidelity, and display decreased ribavirin sensitivity. In addition, these two mutants exhibited significantly increased fitness in vitro. These results demonstrate that recombination and mutation rates are intimately linked. Our results have important implications for understanding the crucial role of the RdRp in virus recombination and fitness, especially in the molecular mechanisms of SVA evolution and pathogenicity.

    IMPORTANCE Recent evidence has emphasized the importance of SVA recombination on virus evolution in vivo. We describe the first assays to study Senecavirus A recombination. Results showed that the RNA-dependent RNA polymerase plays a crucial role in recombination and that recombination can impact fitness of SVA in cell culture. Further, the SVA polymerase fidelity is closely related to the recombination efficiency. The results provide key insights into the role of recombination in positive-strand RNA viruses.

  • Phosphoproteome analysis of cells infected with adapted and non-adapted influenza A virus reveals novel pro- and antiviral signaling networks [Cellular Response to Infection]

  • Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its non-adapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, this data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.

    IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation, on the other hand the host cell triggers anti-viral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This work revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, this work identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.

  • The CCCTC binding factor, CTRL2, modulates heterochromatin deposition and the establishment of HSV-1 latency in vivo [Genome Replication and Regulation of Viral Gene Expression]

  • The cellular insulator protein CTCF plays a role in HSV-1 latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the LAT enhancer was bound by CTCF during latency and underwent CTCF eviction at early times post-reactivation in mice latent with 17syn+. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (CTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells, but not neuronal cells. Following ocular infection, mouse survival decreased in the CTRL2 cohort and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with CTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the ganglia infected with CTRL2 by 31 days post-infection, relative to 17syn+, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, ChIP-seq analyses of TG from CTRL2 infected mice confirmed that the distribution of the repressive H3K27me3 mark on the CTRL2 recombinant genomes was altered compared to the wild-type, indicating that CTRL2 site modulates the repression of IE genes during latency.

    IMPORTANCE It is becoming increasingly clear that chromatin insulators play a key role in the transcriptional control of DNA viruses. The gamma herpesviruses EBV and KSHV utilize chromatin insulators to order protein recruitment and dictate the formation of 3-dimensional DNA loops that spatially control transcription and latency. The contribution of chromatin insulators in alpha herpesvirus transcriptional control is less understood. The work presented here begins to bridge that gap in knowledge by showing how one insulator site in HSV-1 modulates lytic gene transcription and heterochromatin deposition as the HSV-1 genome establishes latency.

  • Adenovirus E1B-55K targets SMARCAL1 for degradation during infection and modulates cellular DNA replication [Virus-Cell Interactions]

  • Here we show that the cellular DNA replication protein and ATR substrate, SMARCAL1, is recruited to viral replication centres early during adenovirus infection and is then targeted in an E1B-55K/E4orf6 and Cullin Ring Ligase-dependent manner for proteasomal degradation. In this regard we have determined that SMARCAL1 is phosphorylated at S123, S129 and S173 early during infection, in an ATR- and CDK- dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centres was shown to be largely dependent upon SMARCAL1 association with the RPA complex, whilst Ad-induced SMARCAL1 phosphorylation also contributed towards SMARCAL1 recruitment to viral replication centres, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1 we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork-speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. We propose therefore, that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.

    Importance

    Viruses have evolved to inhibit cellular DNA damage response pathways that possess anti-viral activities and utilize DNA damage response pathways that possess pro-viral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus regulates, differentially, ATR DNA damage response signalling pathways during infection. The cellular, adenovirus E1B-55K binding protein, E1B-AP5, participates in ATR signalling pathways activated during infection, whilst adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of ATR activator, TOPBP1. The studies detailed herein indicate that adenovirus utilises ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signalling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

  • Seeking closure: How do herpesviruses recruit the cellular ESCRT apparatus? [Minireviews]

  • The Herpesviridae are structurally complex DNA viruses whose capsids undergo primary envelopment at the inner nuclear membrane and secondary envelopment at organelles in the cytoplasm. In both locations there is evidence that envelope formation and scission involve the participation of multiple viral proteins and also the cellular ESCRT apparatus. It nevertheless appears that the best understood viral strategies for ESCRT recruitment, those adopted by the retroviruses and many other families of enveloped RNA viruses, are not utilized by the Herpesviridae, at least during envelopment in the cytoplasm. Thus, although there are a large number of herpesvirus proteins assigned roles in envelopment, there is a dearth of candidates for acquisition of the ESCRT complex and the control of envelope scission. This review summarizes our current understanding of ESCRT association by enveloped viruses, examines what is known of herpesvirus ESCRT utilization in the nucleus and cytoplasm, and identifies candidate cellular and viral proteins that could link enveloping herpesviruses to cellular ESCRT components.

  • Complex membrane remodeling during virion assembly of the 30,000 years-old Mollivirus sibericum [Virus-Cell Interactions]

  • Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary 3D-electron microscopy techniques including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles, never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant and DNA is packed within virions, prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggests that the molecular mechanisms for cellular membrane remodeling and persistence are unique.

    Importance

    Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the World and appear to be unique in several aspects. They belong to at least four viral families and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae" which was isolated from a 30,000 years-old permafrost sample, and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna; (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled; (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding on how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

  • Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets [Pathogenesis and Immunity]

  • Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian aviannndash;origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals, and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.

    Importance

    Subtype H10 avian IAVs have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.

  • Interplay between the epigenetic enzyme lysine (K)-specific demethylase 2B and Epstein-Barr virus infection [Virus-Cell Interactions]

  • The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with EBV-negative sporadic BL cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host-cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B cell transformation. We showed that infection of B cells with EBV leads to downregulation of KDM2B levels. We also showed that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we showed that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host-cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.

    IMPORTANCE. In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, among them the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.

  • The reduced folate carrier: an entry receptor for a novel feline leukemia virus variant [Virus-Cell Interactions]

  • Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, Anndash;D and T, each with distinct receptor usage, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2 phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells, but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Envnndash;receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and the virus replicated in hematopoietic cell lines compared with FeLV-A 61E. These results provide a tool for further investigation of FeLV infectious disease.

    Importance Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirusnndash;receptor interactions under selective pressure by the host results in the emergence of novel viruses.

  • Oral vaccination with replication-competent adenovirus in mice reveals the dissemination of the viral vaccine beyond the gastrointestinal tract [Vaccines and Antiviral Agents]

  • Since the 1970s, replication-competent human adenoviruses 4 and 7 have been used as oral vaccines to protect US soldiers against the severe respiratory diseases caused by these viruses. These vaccines are thought to establish a digestive tract infection conferring protection against respiratory challenge through antibodies. The success of these vaccines makes replication-competent adenoviruses attractive candidates for use as oral vaccine vectors. However, the inability of human adenoviruses to replicate efficiently in laboratory animals has hampered the study of such vectors. Here, we used mouse adenovirus type 1 (MAV-1) in mice to study oral replication-competent adenovirus-based vaccines. We showed that MAV-1 oral administration recapitulates the protection against homologous respiratory challenge observed with adenoviruses 4 and 7 vaccines. Moreover, live oral MAV-1 vaccine better protected against a respiratory challenge than inactivated vaccines. This protection was linked not only with the presence of MAV-1-specific antibodies but also with a better recruitment of effector CD8 T cells. However, unexpectedly, we found that such oral replication-competent vaccine systemically spread all over the body. Our results therefore support using MAV-1 to study replication-competent oral adenovirus-based vaccines but also highlight the fact that those vaccines could disseminate widely in the body.

    IMPORTANCE Replication-competent adenoviruses appear to be promising vectors for the development of oral vaccines in humans. However, study and development of these vaccines suffer from the lack of any reliable animal model. In this study, mouse adenovirus type 1 has been used to develop a small animal model for oral replication-competent adenovirus vaccines. While this model reproduced in mice what is observed with human adenovirus oral vaccines, it also highlighted that oral immunization with such replication-competent vaccine is associated with the systemic spread of the virus. This study is therefore of major importance for the future development of such vaccine platforms and their use in large human populations.

  • Fundamental contribution and host range determination of ANP32A and ANP32B in influenza A virus polymerase activity [Virus-Cell Interactions]

  • The polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase, and to both influence polymerase activity and interspecies restriction. Here we report that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B support the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129-130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32Aaamp;B as key factors for both virus replication and adaption.

    IMPORTANCE The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. Here we provide evidence that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza virus RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily non-functional in supporting viral replication because of a 129-130 site mutation. The 129-130 site plays an important role in ANP32A/B and viral polymerase interaction, therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

  • Phosphorylation of HPV-16 L2 Contributes To Efficient Virus Infectious Entry [Virus-Cell Interactions]

  • The Human Papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are post-translationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and BPV-1 L2 proteins. The phospho-modification of L2, and its presence in HPV pseudovirions (PsVs), was confirmed using anti-phospho L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiency of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.

    IMPORTANCE The Papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However nothing is known about how potential post-translational modifications may affect different aspects of capsid assembly or infectious entry. In this study we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple Papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the Papillomavirus L2 proteins; a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway

  • Nipah and Hendra viral glycoproteins induce comparable homologous but distinct heterologous fusion phenotypes [Virus-Cell Interactions]

  • Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are BSL-4 paramyxoviruses in the growing genus, Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, pathognomonic of henipaviral infections. Here we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (HA tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F/G interactions, supporting the F/G dissociation model of henipaviral-induced membrane fusion, even in the context of heterologous glycoprotein pairs.

    IMPORTANCE The Nipah (NiV) and Hendra (HeV) henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytia formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential co-infections of these or subsequent hosts may alter the outcome of disease.

  • Antagonizing the glucocorticoid receptor impairs explant-induced reactivation in mice latently infected with herpes simplex virus 1 [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) establishes life-long latent infections in neurons. Reactivation from latency can lead to serious recurrent disease, including stromal keratitis, corneal scarring, blindness, and encephalitis. Although numerous studies link stress to an increase in the incidence of reactivation from latency and recurrent disease, the mechanism of action is not well understood. We hypothesized that stress, via corticosteroid mediated activation of the glucocorticoid receptor (GR), stimulates viral gene expression and productive infection during reactivation from latency. Consequently, we tested whether GR activation by the synthetic corticosteroid dexamethasone influenced virus shedding during reactivation from latency using trigeminal ganglia (TG) explants from Swiss Webster mice latently infected with HSV-1, McKrae strain. TG explants from latently infected mice shed significantly higher levels of virus when treated with dexamethasone. Conversely, virus shedding from TG explants was significantly impaired when incubated with media containing the GR-specific antagonist (CORT-108297) or stripped fetal bovine serum, which lacks nuclear hormones and other growth factors. TG explants from latently infected, but not uninfected TG, contained significantly more GR-positive neurons following explant when treated with dexamethasone. Strikingly, VP16 protein expression was detected in TG neurons at 8 hours after explant whereas ICP0 and ICP4 protein expression were not readily detected until 16 hours after explant. Expression of all three viral regulatory proteins was stimulated by dexamethasone. These studies indicated corticosteroid mediated GR activation increased the number of TG neurons expressing viral regulatory proteins, which enhanced virus shedding during explant-induced reactivation from latency.

    IMPORTANCE

    Herpes simplex virus 1 (HSV-1) establishes life-long latent infections in neurons within trigeminal ganglia (TG); periodically reactivation from latency occurs, leading to virus transmission and recurrent disease. Chronic or acute stress increases the frequency of reactivation from latency; how this occurs is not well understood. Herein, we demonstrate the synthetic corticosteroid dexamethasone stimulated explant-induced reactivation from latency. Conversely, a glucocorticoid receptor (GR) antagonist significantly impaired reactivation from latency, indicating GR activation stimulated explant-induced reactivation. The viral regulatory protein, VP16, was readily detected in TG neurons prior to infected cell protein 0 (ICP0) and ICP4 during explant-induced reactivation. Dexamethasone induced expression of all three viral regulatory proteins following TG explant. Whereas the immunosuppressive properties of corticosteroids would facilitate viral spread during reactivation from latency, these studies indicate GR activation increases the number of TG neurons that express viral regulatory proteins during early stages of explant-induced reactivation.

  • Aedes aegypti NeSt1 protein enhances Zika virus pathogenesis by activating neutrophils [Pathogenesis and Immunity]

  • Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase of flavivirus-susceptible cells at the infected bite site. Additionally, an antibody response towards specific salivary gland (SG) components changes the pathogenesis of flavivirus in human populations. To investigate if antigenic SG proteins are capable of enhancing Zika virus (ZIKV) infection, a re-emerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library, and demonstrate a previously undescribed SG protein, we term neutrophil stimulating factor 1 (NeSt1), activates primary mouse neutrophils ex vivo. Passive immunization against NeSt1 decreases pro-IL-1bbeta; and CXCL2 expression, prevents macrophages from infiltrating into the bite site, protects susceptible IFNAR-/-IFNGR-/- (AG129) mice from early ZIKV replication, and ameliorates viral-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenviroment, allowing higher early viral replication and enhancing ZIKV pathogenesis.

    IMPORTANCE When a Zika virus infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identify a molecule in this paper that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We name this molecule neutrophil stimulating factor 1 (NeSt1). When we use antibodies to block NeSt1 in a mouse and then allow Zika virus infected mosquitoes to feed on these mice, they survive much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection.

  • Replication of Marek's Disease Virus is dependent on synthesis of de novo Fatty Acid and Prostaglandin E2 [Virus-Cell Interactions]

  • Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEF). In addition, MDV infected cells contained high levels of fatty acids and showed an increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titre by approximately 30 folds. Addition of the downstream metabolites including malonyl-CoA or palmitic acid completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates COX-2/PGE2 pathway as evident by increased levels of arachidonic acid, COX-2 expression and PGE2 synthesis. Inhibition of COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using shRNA reduced MDV titres, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.

    Importance Disturbances of the lipid metabolism in chickens infected with Marek's disease virus (MDV) contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates fatty acid synthesis (FAS) and induces lipid droplet (LD) formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signalling pathways.

  • Mechanisms of B-cell oncogenesis induced by Epstein-Barr virus [Gem]

  • Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus, which asymptomatically infects majority of the world population. In immune-compromised conditions, EBV can trigger human cancers of epithelial and lymphoid origin. The oncogenic potential of EBV is demonstrated by in-vitro infection and transformation of quiescent B-cells into lymphoblastoid cell lines (LCLs). These cell lines, along with primary infection using genetically engineered viral particles coupled with recent technological advancements have elucidated the underlying mechanisms of EBV-induced B-cell lymphomagenesis.

  • Specific activation of HIV-1 by a bromodomain inhibitor from monocytic cells in humanized mice under ART in vivo [Virus-Cell Interactions]

  • The combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and enables HIV-infected individuals to live long productive lives. However, the persistence of HIV-1 reservoirs of both T and myeloid cells with latent or low-replicating HIV-1 in patients under cART makes HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies to activate and purge these reservoirs. Bromodomain and extraterminal domain proteins (BETs) are epigenetic readers involved in modulating gene expression. Several bromodomain inhibitors (BETi) are reported to activate viral transcription in vitro in HIV-1 latency cell lines in a P-TEFb (CDK9/cyclin T1)-dependent manner. Little is known about the BETi efficacy in activating HIV-1 reservoir cells under cART in vivo. In this study, we report that a BETi (I-BET151) efficiently activated HIV-1 reservoirs under effective cART in humanized mice in vivo. Interestingly, I-BET151 during suppressive cART in vivo activated HIV-1 gene expression only in monocytic cells, but not in CD4+ T cells. We further demonstrate that BETi preferentially enhanced HIV-1 gene expression in monocytic cells than in T cells and, whereas CDK9 was involved in activating HIV-1 by I-BET151 in both monocytic and T cells, CDK2 enhanced HIV-1 transcription in monocytic cells but inhibited it in T cells. Our findings reveal a role of CDK2 in differential modulation of HIV-1 gene expression in myeloid cells and in T cells, and provides a novel strategy to reactivate monocytic reservoirs with BETi during cART.

    IMPORTANCE Bromodomain inhibitors have been reported to activate HIV-1 transcription in vitro but their effect on activation of HIV-1 reservoirs during cART in vivo is unclear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells in cART-treated mice. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in HIV-1 infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells, and a combination of reservoir activation agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

  • Characterizing a murine model for astrovirus using viral isolates from persistently infected immunocompromised mice [Pathogenesis and Immunity]

  • Human astroviruses are single-stranded enteric RNA viruses that cause a spectrum of disease ranging from asymptomatic infection to systemic extra-gastrointestinal spread; however, they are among the least characterized enteric viruses and there is a lack of a well-characterized small animal model. Finding that immunocompromised mice were resistant to human astrovirus infection via multiple routes of inoculation, our studies aimed to determine whether murine astrovirus (MuAstV) could be used to model human astrovirus disease. We experimentally infected wildtype mice with MuAstV isolated from immunocompromised mice and found that the virus was detected throughout the gastrointestinal tract, including the stomach, but was not associated with diarrhea. The virus was also detected in the lung. Although virus levels were higher in recently weaned mice, the levels were similar in male and female adult mice. Using two distinct viruses isolated from different immunocompromised mouse strains, we observed virus strainnndash;specific differences in the duration of infection (3 vs. 10 weeks) in wildtype mice, indicating that the within-host immune pressure from donor mice shaped the virus kinetics in immunocompetent recipient hosts. Both virus strains elicited minimal pathology and a lack of sustained immunity. In summary, MuAstV represents a useful model for studying asymptomatic human infection and gaining insight to the astrovirus pathogenesis and immunity.

    IMPORTANCE: Astroviruses are widespread in both birds and mammals; however, they are among the least characterized enteric viruses and little is known about the pathogenesis and the immune response to the virus due to the lack of a small animal model. Herein, we describe two distinct strains of murine astrovirus that cause infections in immunocompetent mice that mirror aspects of asymptomatic human infections, including minimal pathology and short-lived immunity. However, we noted that the duration of infection differed greatly between the strains, highlighting an important facet of these viruses that was not previously appreciated. Given the ubiquitous nature and diversity of murine astroviruses coupled with the continuous likelihood of re-infection raises the possibility of viral interference with other mouse models of disease.

  • Human papillomavirus type 11 early protein E6 activates autophagy by repressing AKT/mTOR and Erk/mTOR [Virus-Cell Interactions]

  • Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which is a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV type 11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-GFP-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knockdown 11E6 and showed that these effects require continued 11E6 expression. Compared with the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of decreased phosphorylation levels of the canonical autophagy repressor mTOR at its Ser2448 position (mTORC1 phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.

    Importance: We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.

  • Characterization of Novel Reoviruses [Wad Medani virus (Orbivirus) and Kundal (Coltivirus)] collected from Hyalomma antolicum ticks in India during CCHF surveillance [Genetic Diversity and Evolution]

  • In 2011, ticks were collected from livestock following an outbreak of Crimean Congo Hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV) respectively. Traditional RT-PCR identification of known viruses was unsuccessful, but a next-generation sequencing approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus, and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identity, KUNDV is proposed to be a new species of Coltivirus. Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other Orbi- and Colti- viruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.

    Importance Ticks, mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammanvanpettai virus (KVPTV) and with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo Hemorrhagic Fever virus, Babesia, Theileria and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of these KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

  • Reconstruction of the genetic history and the current spread of HIV-1 subtype A in Germany [Genetic Diversity and Evolution]

  • HIV-1 non-B infections have been increasing in Europe for several years. In Germany, subtype A belongs to the most abundant non-B subtypes showing an increasing prevalence of 8.3% among new infections in 2016. Here we trace the origin and examine the current spread of the German HIV-1 subtype A epidemic. Bayesian coalescence and birth-death analyses were performed with 180 German HIV-1 pol-sequences and 528 related and publicly available sequences to reconstruct the population dynamics and fluctuations for each of the transmission groups.

    Our reconstructions indicate two distinct sources of the German subtype A epidemic, with an Eastern European and an Eastern African lineage both co-circulating in the country. A total of 13 German-origin clusters were identified; among these, 6 clusters showed recent activity. Introductions leading to further countrywide spread originated predominantly from Eastern Africa when introduced before 2005. Since 2005 however, spreading introductions have occurred exclusively within the Eastern European clade. Moreover, we observed changes in the main route of subtype A transmission. The beginning of the German epidemic (1985-1995) was dominated by heterosexual transmission of the Eastern African lineage. Since 2005 transmissions among German MSM are increasing and are associated with the Eastern European lineage. Infections among people who inject drugs dominated between 1998 and 2005.

    Our findings on HIV-1 subtype A infections provide new insights into the spread of this virus and extend the understanding of the HIV epidemic in Germany.

    Importance HIV-1 subtype A is the second most prevalent subtype worldwide, with a high prevalence in Eastern Africa and Eastern Europe. However, an increase of non-B infections including subtype A infections has been observed in Germany and other European countries. There has simultaneously been an increased flow of refugees into Europe and especially into Germany, raising the question of whether the surge in non-B infections resulted from this increased immigration or whether German transmission chains are mainly involved. This study is the first comprehensive subtype A study from a western European country analyzing in detail its phylogenetic origin, the impact of various transmission routes and its current spread. The results and conclusions presented provide new and substantial insights for virologists, epidemiologists, and the general public health sector. In this regard, they should be useful to those authorities responsible for developing public health intervention strategies to combat the further spread of HIV/AIDS.

  • Zika Virus Subverts Stress Granules to Promote and Restrict Viral Gene Expression [Virus-Cell Interactions]

  • Flaviviruses limit the cell stress response by preventing the formation of stress granules and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific siRNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR and YB1. Knock-down of TIA-1 and TIAR affected ZIKV protein and RNA levels, but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection, while HuR exhibits an antiviral effect. The consequence of ZIKV re-localizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis.

    Importance: Many viruses inhibit stress granules (SGs). In this study, we observed that ZIKV restricts SG assembly likely by re-localizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this re-emerging arbovirus.

  • Arterivirus nsp4 antagonizes interferon-{beta} production by proteolytically cleaving NEMO at multiple sites [Virus-Cell Interactions]

  • Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon (IFN)-bbeta; production by cleaving the NF-B essential modulator (NEMO) at a single site, glutamate 349 (E349). Herein, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-bbeta; production by targeting NEMO for proteolytic cleavage, and that the scission occurred at four sites- E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, the scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-bbeta; production, we serendipitously found that a NEMO fragment (1nndash;349) could activate IFN-bbeta; transcription more robustly than full-length NEMO whereas all other NEMO cleavage products were abrogated for the IFN-bbeta;-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.

    IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrated that both EAV and PRRSV nsp4 cleave NEMO at multiple sites, and that this strategy is essential for disruption of type I IFN production. Moreover, we revealed that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has only been observed with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro. In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

  • C19ORF66 broadly escapes viral-induced endonuclease cleavage and restricts Kaposi's Sarcoma Associated Herpesvirus (KSHV) [Cellular Response to Infection]

  • One striking characteristic of certain herpesviruses is their ability to induce rapid and widespread RNA decay in order to gain access to host resources. This phenotype is induced by viral endoribonucleases, including SOX in KSHV, muSOX in MHV68, BGLF5 in EBV and vhs in HSV-1. Here, we performed comparative RNA-seq upon expression of these herpesviral endonucleases in order to characterize their effect on the host transcriptome. Consistent with previous reports, we found that approximately two thirds of transcripts are downregulated in cells expressing any of these viral endonucleases. Among transcripts spared from degradation, we uncovered a cluster of transcripts that systematically escape degradation from all tested endonucleases. Among these escapees, we identified C19ORF66 and reveal that this transcript is protected from degradation by its 3'UTR. We then show that C19ORF66 is a potent KSHV restriction factor by impeding early viral gene expression, suggesting that its ability to escape viral cleavage may be an important component of the host response to viral infection. Collectively, our comparative approach is a powerful tool to pinpoint key regulators of the viral-host interplay and led us to uncover a novel KSHV regulator.

    Importance Viruses are master regulator of the host gene expression machinery. This is crucial to promote viral infection and to dampen host immune responses. Many viruses, including herpesviruses express RNAses that reduce host gene expression through widespread mRNA decay. However, it emerged that some mRNAs escape this fate, although it has been difficult to determine whether these escaping transcripts benefit viral infection or instead participate in an anti-viral mechanism. To tackle this question, we compared the effect of the herpesviral RNAses on the human transcriptome and identified a cluster of transcripts consistently escaping degradation from all tested endonucleases. Among the protected mRNAs, we identified the transcript C19ORF66 and showed that it restricts KSHV infection. Collectively these results provide a framework to explore how the control of RNA fate in the context of viral-induced widespread mRNA degradation may influence the outcome of viral infection.

  • Development of Influenza B Universal Vaccine Candidates using the "Mosaic" Hemagglutinin Approach [Vaccines and Antiviral Agents]

  • Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. However, protection by current seasonal vaccines is suboptimal due to the antigenic changes of the circulating strains. In this study, we report a novel universal influenza B virus vaccination strategy based on mosaic hemagglutinins. We generated mosaic B hemagglutinins by replacing the major antigenic sites of the type B hemagglutinin with corresponding sequences from exotic influenza A hemagglutinins and expressed them as soluble trimeric proteins. Sequential vaccination with recombinant mosaic B hemagglutinin proteins conferred cross-protection against both homologous and heterologous influenza B virus strains in the mouse model. Of note, we rescued recombinant influenza B viruses expressing mosaic B hemagglutinins, which could serve as the basis for a universal influenza B virus vaccine.

    IMPORTANCE This work reports a universal influenza B virus vaccination strategy based on focusing antibody responses to conserved head and stalk epitopes of the hemagglutinin. Recombinant mosaic influenza B hemagglutinin proteins and recombinant viruses have been generated as novel vaccine candidates. This vaccine strategy provided broad cross-protection in the mouse model. Our findings will inform and drive the development toward a more effective influenza B virus vaccine.

  • Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function [Structure and Assembly]

  • Most broadly neutralizing antibodies and many entry inhibitors target the pre-triggered (State-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two published Env mutants designed to stabilize this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some State-1-preferring ligands. By contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is non-functional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.

    IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Env) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called State 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into State 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the State-1 conformation.

  • Envelope protein glycosylation mediates Zika virus pathogenesis [Pathogenesis and Immunity]

  • Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barreeacute; syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154) but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice. In contrast, non-glycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously, but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing DC-SIGN and DC-SIGNR, suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of non-glycosylated ZIKV in mice.

    IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barreeacute; syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated non-glycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that non-glycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

  • Histone deacetylase inhibitor SAHA suppresses human adenovirus gene expression and replication [Vaccines and Antiviral Agents]

  • Human adenovirus (HAdV) causes minor illnesses in most patients, but can lead to severe disease and death in pediatric, geriatric and immunocompromised individuals. No approved antiviral therapy currently exists for the treatment of these severe HAdV-induced diseases. In this study, we show that the pan-histone deacetylase (HDAC) inhibitor SAHA reduces HAdV-5 gene expression and DNA replication in tissue culture, ultimately decreasing virus yield from infected cells. Importantly, SAHA also reduced gene expression from more virulent and clinically relevant serotypes including HAdV-4 and HAdV-7. In addition to SAHA, several other HDAC inhibitors (e.g. TSA, apicidin, panobinostat) also affected HAdV gene expression. We determined that loss of class I HDAC activity, mainly HDAC2, impairs efficient expression of viral genes, and that E1A physically interacts with HDAC2. Our results suggest that HDAC activity is necessary for HAdV replication, which may represent a novel pharmacological target in HAdV-induced disease.

    IMPORTANCE Although human adenovirus (HAdV) can cause severe diseases that can be fatal in some populations, there are no effective treatments to combat HAdV infection. In this study, we determined that the pan-histone deacetylase (HDAC) inhibitor SAHA has inhibitory activity against several clinically relevant serotypes of HAdV. This FDA-approved compound affects various stages of the virus lifecycle, and reduces virus yield even at low concentrations. We further report that Class I HDAC activity, particularly HDAC2, is required for efficient expression of viral genes during lytic infection. Investigation of the mechanism underlying SAHA-mediated suppression of HAdV gene expression and replication will enhance current knowledge of virus-cell interaction, and may aid in the development of more effective antivirals with lower toxicity for the treatment of HAdV infections.

  • African swine fever virus Armenia/07 virulent strain controls IFN-{beta} production through cGAS-STING pathway [Virus-Cell Interactions]

  • African swine fever virus (ASFV) is a complex, cytoplasmic dsDNA virus, currently expanding throughout the world. Currently circulating virulent genotype II Armenia/07-like viruses causes fatal disease in pigs and wild boar, whereas attenuated strains induce infections with varying levels of chronic illness.

    Sensing cytosolic dsDNA, mainly by the key DNA sensor cGAS, leads to the synthesis of type I interferon, and involves signaling through STING, TBK1 and IRF3. After phosphorylation, STING translocates from the ER to the Golgi and to the perinuclear region, therefore being an indispensable adaptor connecting the cytosolic detection of DNA to theTBK1-IRF3 signaling pathway.

    We demonstrate that attenuated NH/P68, but not virulent Armenia/07, activates the cGAS-STING-IRF3 cascade very early during infection, inducing STING phosphorylation and trafficking through a mechanism involving cGAMP. Both TBK1 and IRF3 are subsequently activated, and in response to this, high level of IFN-bbeta; production was produced during NH/P68 infection; in contrast, Armenia/07 infection generated IFN-bbeta; levels below those of uninfected cells. Our results show that virulent Armenia/07 ASFV controls the cGAS-STING pathway, but these mechanisms are not at play when porcine macrophages are infected with the attenuated NH/P68 ASFV.

    These findings show for the first time the involvement of the cGAS-STING-IRF3 route in ASFV infection, where IFN-bbeta; production or inhibition was found after infection by attenuated or virulent ASFV strains, respectively, thus reinforcing the idea that ASFV virulence vs. attenuation may be a phenomenon grounded in ASFV-mediated innate immune modulation where the cGAS-STING pathway might play an important role.

    IMPORTANCE African swine fever, a devastating disease for domestic pigs and wild boar is currently spreading in Europe, Russia and China, becoming a global threat with huge economic and ecological consequences. One interesting aspect of ASFV biology is the molecular mechanism leading to high virulence of some strains compared to more attenuated strains, which produce subclinical infections. In this work, we show that the presently circulating virulent Armenia/07 virus blocks the synthesis of IFN-bbeta;, a key mediator between the innate and adaptive immune response. Armenia/07 inhibits the cGAS-STING pathway by impairing STING activation during infection. In contrast, the cGAS-STING pathway is efficiently activated during NH/P68 attenuated strain infection, leading to the production of high amounts of IFN-bbeta;. Our results show for the first time the relationship between the cGAS-STING pathway and ASFV virulence, contributing to uncover the molecular mechanisms of ASFV virulence and to the rational development of ASFV vaccines.

  • Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System [Pathogenesis and Immunity]

  • Central nervous system (CNS) disease is one of the most common extra-respiratory tract complication of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic viruses. Little is known about the interaction between influenza A viruses and cells of the CNS, and therefore it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH), astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells which was associated with efficient attachment and infection. Seasonal H3N2 virus and to a lesser extent pandemic H1N1 replicated trypsin-dependent in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multi-basic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate replication. Taken together, we showed that the MBCS and to a lesser extent ability to attach and are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative HA cleavage mechanism and preference for aalpha;-2,3 linked sialic acids allowing efficient attachment, contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.

    IMPORTANCE Central nervous system (CNS) disease is one of the most common extra-respiratory tract complications of influenza A virus infections and frequency and severity differ between seasonal, pandemic and zoonotic viruses. However, little is known about the interaction of these viruses with cells of CNS. Differences among seasonal, pandemic and zoonotic viruses in replication efficacy and cleavability in CNS cells partially explain the higher frequency and severity of zoonotic viruses. Identifying important viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.

  • Modeling HIV-1 latency using primary CD4+ T cells from HIV-1 infected ART suppressed individuals [Virus-Cell Interactions]

  • The small frequency of latently HIV-infected cells in vivo limits the testing of potential HIV cure strategies using cells from successfully suppressed individuals. To date, primary cell models of latency use cells infected in vitro. Primary CD4+ T cell models carrying an individual's endogenous HIV reservoir that recapitulate in vivo conditions of HIV latency are still outstanding. We developed a primary CD4+ T cell model of HIV latency derived from memory CD4+ T cells isolated from virally suppressed HIV-infected individuals, that recapitulates HIV-1 latency and viral reactivation events. This model is based on the expansion of primary CD4+ T cells up to 300 fold in cell number. These cells reestablish a resting state without active virus production after extended culture and maintain a stable number of total HIV proviruses. The ability of these cells to respond to various classes of latency-reversing agents is similar to ex vivo CD4+ T cells directly isolated from blood. Importantly, viral outgrowth assays confirmed the ability of these expanded cells to produce replication competent endogenous virus. In sum, this model recapitulates ex vivo viral reactivation conditions, captures the variability between individuals with different HIV reservoir and provides large numbers of cells for testing multiple agents from a single donor. The use of this novel model will allow accurate exploration of novel cure approaches, either aimed at promoting viral reactivation or maintenance of sustained latency.

    Importance: Primary cell models of HIV latency have been very useful to identify mechanisms contributing to HIV latency and to evaluate potential HIV cure strategies. However, the current models utilize in vitro infection with exogenous virus that do not fully recapitulate virus reactivation profiles of endogenous HIV in in vivo infected CD4+ T cells. In contrast, CD4+ T cells from HIV-infected individuals require leukapheresis to obtain sufficient amounts of cells to interrogate the HIV reservoir in vitro. In the model we propose here, in vitro expansion and extended culture of primary CD4+ T cells isolated from virally suppressed HIV-infected individuals enable obtaining large numbers of cells harboring endogenous latent HIV reservoir without performing leukaphereses. This model captures the variability of HIV reservoir seeded in different individuals and should be useful to evaluate future HIV cure strategies.

  • Analysis of coronavirus temperature-sensitive mutants reveals an interplay between the macrodomain and papain-like protease impacting replication and pathogenesis [Genome Replication and Regulation of Viral Gene Expression]

  • Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain and the papain-like protease 2 (PLP2) domain of nonstructural protein 3, a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contribution of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency of plating analysis performed at permissive and non-permissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Co-immunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance the proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the non-permissive temperature, as compared to the wild-type proteins. Importantly, we show that the macrodomain- and PLP2-mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.

    IMPORTANCE Coronaviruses are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control these infections in humans. Viral macrodomains, enzymes that remove post-translational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating (DUB) activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

  • Identification and characterization of a human coronavirus 229E nonstructural protein 8-associated RNA 3'-terminal adenylyltransferase activity [Genome Replication and Regulation of Viral Gene Expression]

  • Coronavirus nonstructural protein (nsp) 8 has been suggested to have diverse activities, including llsquo;noncanonicalrrsquo; template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs, respectively. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Substitution of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a non-viable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3'-polyadenylation of viral plus-strand RNAs.

    IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies suggest an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce complementary RNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

  • Interferon Gamma Inhibits Varicella-Zoster Virus Replication in a Cell Line-Dependent Manner [Genome Replication and Regulation of Viral Gene Expression]

  • The major immediate-early IE62 protein of varicella-zoster virus (VZV) is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate-early (IE), early (E), and late (L) genes. Interferon gamma (IFN-) is a potent cytokine produced following primary VZV infection. Furthermore, VZV reactivation correlates with a decline in IFN--producing immune cells. Our results showed that treatment with 20 ng/ml of IFN- completely reduced intracellular VZV yield in lung epithelial A549, lung fibroblast MRC-5, and retinal epithelial ARPE-19 cells at 4 days post-VZV infection. However, IFN- reduced virus yield by only 2-fold in melanoma MeWo cells compared to that of untreated cells. IFN-bbeta; significantly inhibited VZV replication in both ARPE-19 and MeWo cells. In luciferase assays with VZV ORF61 promoter reporter plasmid, IFN- abrogated the trans-activation activity of IE62 by 95%, 97%, and 89% in A549, ARPE-19, and MRC-5 cells, respectively. However, IFN- abrogated IE62's trans-activation activity by 16% in MeWo cells, indicating that IFN- inhibits VZV replication as well as IE62-mediated trans-activation in a cell line-dependent manner. The expression of VZV IE62 and ORF63 suppressed by IFN- was restored by JAK1 inhibitor treatment, indicating that the inhibition of VZV replication is mediated by JAK/STAT1 signaling. In the presence of IFN-, knockdown of interferon response factor 1 (IRF1) increased VZV replication. Ectopic expression of IRF1 reduced VZV yields by 4,000-fold in MRC-5 and ARPE-19 cells, but by 3-fold in MeWo. These results suggest that IFN- blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner.

    IMPORTANCE Our results showed that IFN- significantly inhibited VZV replication in a cell line-dependent manner. IFN- inhibited VZV gene expression after the immediate-early stage of infection and abrogated IE62-mediated trans-activation. These results suggest that IFN- blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner. Understanding the mechanisms by which IFN- plays a role in VZV gene programming may be important in determining the tissue restriction of VZV.

  • Oxazole-benzenesulfonamide derivatives inhibit HIV-1 reverse transcriptase interaction with cellular eEF1A and reduce viral replication [Vaccines and Antiviral Agents]

  • HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription and replication, indicating its potential as an anti-HIV-1 target. Here we develop a sensitive, live-cell split luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interacting with eEF1A. We used this to screen a small molecule library and discovered small molecule oxazole-benzenesulfonamides (C7, C8, C9), which dose-dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer-interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay, but did inhibit reverse transcription and infection of both wild type (WT) and NNRTI-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides and the antiviral activity was most potent against replication stages before 8 h post-infection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days post-infection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.

    IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

  • Identification of CP77 as the third orthopoxvirus SAMD9 and SAMD9L inhibitor with a unique specificity for a rodent SAMD9L [Virus-Cell Interactions]

  • Orthopoxviruses (OPXVs) have a broad host range in mammalian cells, but Chinese hamster ovary (CHO) cells are non-permissive for vaccinia virus (VACV). Here, we revealed a species-specific difference in host restriction factor SAMD9L as the cause for the restriction and identified orthopoxvirus CP77 as a unique inhibitor capable of antagonizing Chinese hamster SAMD9L (chSAMD9L). Two known VACV inhibitors of SAMD9 and SAMD9L (SAMD9aamp;L);, K1 and C7, can bind human and mouse SAMD9aamp;L;, but neither can bind chSAMD9L. CRISPR-Cas9 knockout of chSAMD9L from CHO cells removed the restriction for VACV, while ectopic expression of chSAMD9L imposed the restriction for VACV in a human cell line, demonstrating that chSAMD9L is a potent restriction factor for VACV. Contrary to K1 and C7, cowpox virus CP77 can bind chSAMD9L and rescue VACV replication in cells expressing chSAMD9L, indicating that CP77 is yet another SAMD9L inhibitor but has a unique specificity for chSAMD9L. Binding studies showed that the N-terminal 382 amino acids of CP77 were sufficient for binding chSAMD9L and that both K1 and CP77 target a common internal region of SAMD9L. Growth studies with nearly all OPXV species showed that the ability of OPXVs in antagonizing chSAMD9L correlates with CP77 gene status and that a functional CP77 ortholog was maintained in many OPXVs, including monkeypox virus. Our data suggest that species-specific difference in rodent SAMD9L poses a barrier for cross-species OPXV infection and that OPXVs have evolved three SAMD9aamp;L inhibitors with different specificities to overcome this barrier.

    IMPORTANCE: Several OPXV species, including monkeypox virus and cowpox virus, cause zoonotic infection in humans. They are believed to use wild rodents as the reservoir or intermediate hosts, but the host or viral factors that are important for OPXV host range in rodents are unknown. Here, we showed that the abortive replication of several OPXV species in a Chinese hamster cell line was caused by a species-specific difference in the host antiviral factor SAMD9L, suggesting that SAMD9L divergence in different rodent species poses a barrier for cross-species OPXV infection. While the Chinese hamster SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human and mouse SAMD9aamp;L;, it can be inhibited by cowpox virus CP77, indicating that OPXVs encode three SAMD9aamp;L inhibitors with different specificity. Our data suggest that OPXV host range in broad rodent species depends on three SAMD9aamp;L inhibitors with different specificities.

  • By binding CD80 and CD86, the vaccinia virus' M2 protein blocks their interactions with both CD28 and CTLA4 and potentiates CD80's binding to PD-L1 [Virus-Cell Interactions]

  • In this article we report that the M2 protein encoded by the vaccinia virus is secreted as a homo-oligomer by infected cells and binds two central co-stimulation molecules: CD80 (B7-1) and CD86 (B7-2). These interactions block the ligation of the two B7 proteins to both soluble CD28 and soluble CTLA4, but favor the binding of soluble PD-L1 to soluble CD80. M2L gene orthologues are found in several other poxviruses and the B7/CD28-CTLA4 blocking activity has been identified for several culture supernatants of orthopoxvirus infected cells and for recombinant myxoma virus M2 protein homolog (i. e. Gp120LP). Overall, these data indicate that the M2 poxvirus family of proteins may be involved in immunosuppressive activities broader than the NF-kB inhibition already reported (Gedey et al. 2006). A Copenhagen vaccinia virus deleted for the non-essential M2L locus was generated and compared to with its parental virus. This M2L deleted vaccinia virus, unlike the parental virus, does not generate interference with the B7/CD28-CTLA4-PD-L1 interactions. Moreover, this deletion did not affect any key features of the virus (in vitro replication, oncolytic activities in vitro and in vivo, and intratumoral expression of a transgene in an immunocompetent murine model). Altogether, these first results suggest that the M2 protein has the potential to be used as a new immunosuppressive biotherapeutic, and that the M2L-deleted Vaccinia virus represents an attractive new oncolytic platform with an improved immunological profile.

    IMPORTANCE The vaccinia virus encodes in its genome several genes dedicated to the inhibition of host immune response. Among them M2L was reported to inhibit the intracellular NF-kB pathway. We report here several new putative immunosuppressive activities of M2 protein. M2 protein is secreted and binds cornerstone costimulatory molecules (CD80/CD86). M2 binding to CD80/CD86, blocks their interaction with soluble CD28/CTLA4, but also favors the soluble PD-L1/CD80 association. These findings open the way for new investigations deciphering the immune system effects of soluble M2 protein. Moreover, a vaccinia virus deleted of its M2L has been generated and characterized as a new oncolytic platform. The replication and oncolytic activities of the M2L-deleted vaccinia virus are indistinguishable from those of the parental virus. More investigations are needed to characterize in detail the immune response triggered against both the tumor and the virus by this M2 defective vaccinia virus.

  • Voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) act as functional receptors for lymphocystis disease virus infection [Virus-Cell Interactions]

  • In previous researches a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was identified as putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32 kDa viral attachment protein (VAP) of LCDV, monoclonal antibodies (MAbs) against 27.8R and 32 kDa VAP were developed. In this study, voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder were confirmed in accordance with the 27.8R. The recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1 and the 27.8 kDa protein in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Pre-incubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies could significantly decrease the percentages of LCDV-infected cells and LCDV copy numbers, block virus infection and delay the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were up-regulated to the maximum level at 12 h and 48 h post LCDV infection respectively. VDAC2/RACK1 knockdown through siRNA could significantly reduce VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls; while VDAC2/RACK1 expression on LCDV non-permissive epithelial papillosum cells (EPC) conferred susceptibility to LCDV infection, indicating VDAC2 and RACK1 was sufficient to allow LCDV entry and infection. All these results collectively evidenced that VDAC2 and RACK1 functioned as receptors for LCDV entry and infection.

    IMPORTANCE Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying LCDV-host interaction remains unclear. Here, the voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) were confirmed in accordance with the 27.8 kDa putative cellular receptor for LCDV, and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and they were functional receptors that initiated LCDV infection for the first time, which led to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interaction.

  • Intra-host Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract [Genetic Diversity and Evolution]

  • Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10-70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intra-host microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. By contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection period, extensive genome-wide purifying selection shaped variant diversity in stallion reproductive tract. Overall, the non-stochastic nature of EAV evolution during persistent infection was driven by active intra-host selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5 and the nsp2 encoding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intra-host evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV, identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.

    IMPORTANCE EAV can persist in the reproductive tract of infected stallions and, consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens is the primary site of viral persistence in the stallion reproductive tract, and is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intra-host evolution of EAV during acute and persistent infection and demonstrates that persistent infection is characterized by extensive genome-wide purifying selection and a non-stochastic evolutionary pattern mediated by intra-host selective pressure, with important nucleotide substitutions occurring in ORFs 1a (region encoding nsp2), 3 and 5.

  • High-throughput Screening and Identification of Potent Broad-spectrum Inhibitors of Coronaviruses [Vaccines and Antiviral Agents]

  • Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro. We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofeti, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus-entry assays, and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.

    IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro. Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

  • Glucosylceramidase Maintains Influenza Infection By Regulating Endocytosis [Virus-Cell Interactions]

  • Influenza is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza infection depends on cellular lipids including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza infection following CRISPR/Cas9-mediated knockout of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) GFP reporter virus. We further showed that reduction in infection correlated with impaired influenza trafficking to late endosomes, and hence fusion, and entry. To examine whether GBA is required for other enveloped viruses, we compared entry mediated by the glycoproteins of Ebola, influenza, vesicular stomatitis, and measles viruses in GBA knockout cells. Entry inhibition was relatively robust for Ebola and influenza, modest for VSV, and mild for measles, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested and found that trafficking of epidermal growth factor to late endosomes, as well as degradation of its receptor, were impaired in GBA knockout cells. Collectively our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as cellular cargos including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize llsquo;late penetratingrrsquo; viruses, including influenza.

    IMPORTANCE Influenza is a viral pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza enters host cells may lead to more efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza trafficking to late endosomes and consequent fusion, entry, and infection. We also provide evidence that promotion of influenza entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos such as the EGF receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as pathogenic processes including virus entry and Gaucher disease.

  • Chicken organic anion transporting polypeptide 1A2, a novel avian hepatitis E virus (HEV) ORF2-interacting protein, is involved in avian HEV infection [Virus-Cell Interactions]

  • Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion transporting polypeptide 1A2 (OATP1A2) from the chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by the GST pull-down assay. Then, the GST pull-down and indirect ELISA assays further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo. Finally, when the functions of OATP1A2 in the cells are inhibited by its substrates, an inhibitor or blocked by ap237 or anti-OATP1A2 sera, the attachment and infection of avian HEV of the host cells is significantly reduced. Collectively, these results displayed that for the first time, OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provided new insight to understand the process of avian HEV infection of host cells.

    IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened from chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in LMH cells can promote ap237 adsorption as well as avian HEV adsorption and infection of LMH cells. When the function of OATP1A2 in the cells was inhibited by substrates or inhibitors, the attachment and infection of avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with the tissues of avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

  • Sequence determinants in gammaretroviral Env cytoplasmic tails dictate virus-specific pseudotyping compatibility [Structure and Assembly]

  • Viruses can incorporate foreign glycoproteins to form infectious particles through a process known as pseudotyping. However, not all glycoproteins are compatible with all viruses. Despite the fact that viral pseudotyping is widely used, what makes a virus/glycoprotein pair compatible is poorly understood. To study this, we chose to analyze a gammaretroviral glycoprotein (Env) whose compatibility with different viruses could be modulated through small changes in its cytoplasmic tail (CT). One form of this glycoprotein is compatible with Murine Leukemia Virus (MLV) particles but incompatible with Human Immunodeficiency Virus 1 (HIV-1) particles, while the second is compatible with HIV-1 particles but not with MLV particles. To decipher the factors affecting virus-specific Env incompatibility, we characterized Env incorporation, maturation, cell-to-cell fusogenicity, and virus-to-cell fusogenicity of each Env. The HIV-1 particle incompatibility correlated with less efficient cleavage of the R-peptide by HIV-1 protease. However, the MLV particle incompatibility was more nuanced. MLV incompatibility appeared to be caused by lack of incorporation into particles, yet incorporation could be restored by further truncating the CT or by using a chimeric MLV Gag containing HIV-1 MA, without fully restoring infectivity. The MLV particle incompatibility appeared to be caused in part by fusogenic repression in MLV particles through an unknown mechanism. This study demonstrates that the Env CT can dictate functionality of Env within particles in a virus-specific manner.

    IMPORTANCE Viruses utilize viral glycoproteins to efficiently enter target cells during infection. How viruses acquire viral glycoproteins has been studied to understand the pathogenesis of viruses and develop safer and more efficient viral vectors for gene therapies. The CTs of viral glycoproteins have been shown to regulate various stages of the glycoprotein biogenesis, but a gap still remains in understanding the molecular mechanism of glycoprotein acquisition and functionality regarding the CT. Here we studied the mechanism of how specific mutations in the CT of a gammaretroviral envelope glycoprotein distinctly affect infectivity with two different viruses. Different mutations caused failure of glycoproteins to function in a virus-specific manner due to distinct fusion defects, suggesting that there are virus-specific characteristics affecting glycoprotein functionality.

  • The membrane-spanning peptide and acidic cluster dileucine sorting motif of UL138 are required to downregulate MRP1 drug transporter function in human cytomegalovirus infected cells. [Virus-Cell Interactions]

  • The human cytomegalovirus (HCMV) UL138 protein downregulates the cell surface expression of the multidrug resistance-associated protein 1 (MRP1) transporter. We examined the genetic requirements within UL138 for MRP1 downregulation. We determined the acidic cluster dileucine motif is essential for UL138 mediated downregulation of MRP1 steady state levels and inhibition of MRP1 efflux activity. We also discovered that the naturally-occurring UL138 protein isoforms, full-length long UL138 and a short isoform missing the N-terminal membrane spanning domain, have different abilities to inhibit MRP1 function. Cells expressing the long isoform of UL138 show decreased MRP1 steady state levels and fail to efflux an MRP1 substrate. Cells expressing the short isoform of UL138 also decreased MRP1 levels, but not to the same magnitude, and they continue to efficiently efflux an MRP1 substrate. Thus, the membrane spanning domain, while dispensable for a UL138-mediated decrease in MRP1 protein levels, is necessary for a functional inhibition of MRP1 activity. Our work defines the genetic requirements for UL138-mediated MRP1 downregulation and anticipates viral escape mutants that may evolve during therapies targeting this function of UL138.

    IMPORTANCE HCMV UL138 curtails the activity of the MRP1 drug transporter by reducing its steady state levels, leaving cells susceptible to killing by cytotoxic agents normally exported by MRP1. It has been suggested in the literature that capitalizing on this UL138-induced vulnerability could be a potential antiviral strategy against virally infected cells, particularly those harboring a latent infection during which UL138 is one of the few viral proteins expressed. Therefore, identifying the regions of UL138 required for MRP1 downregulation and predicting genetic variants that may be selected upon UL138-targeted chemotherapy are important ventures. Here we present the first structure-function examination of UL138 activity and determine that its transmembrane domain and acidic cluster dileucine Golgi sorting motif are required for functional MRP1 downregulation.

  • Atovaquone inhibits arbovirus replication through the depletion of intracellular nucleotides [Vaccines and Antiviral Agents]

  • Arthropod-borne viruses represent a significant public health threat worldwide yet there are few antiviral therapies or prophylaxis targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA approved drugs. In this study, we addressed the antiviral role of atovaquone, a FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides indicating that atovaquone is functioning through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children.

    IMPORTANCE The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-acceptable drugs that have the potential to mitigate arbovirus infections and disease yet this has not been explored in detail. In this study, we show that the common antiparasitic drug, atovaquone, inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.

  • Modulation of sialic acid dependence influences the CNS transduction profile of adeno-associated viruses [Virus-Cell Interactions]

  • Central nervous system (CNS) transduction by systemically administered recombinant adeno-associated viral (AAV) vectors requires crossing the blood brain barrier (BBB). We recently mapped a structural footprint on the AAVrh.10 capsid, which when grafted onto the AAV1 capsid (AAV1RX) enables viral transport across the BBB; however, the underlying mechanisms remain unknown. Here, we establish through structural modeling that this footprint overlaps in part with the sialic acid (SIA) footprint on AAV1. We hypothesized that altered SIA-capsid interactions may influence the ability of AAV1RX to transduce the CNS. Using AAV1 variants with altered SIA footprints, we map functional attributes of these capsids to their relative SIA dependence. Specifically, capsids with ablated SIA binding can penetrate and transduce the CNS with low-to-moderate efficiency. In contrast, AAV1 shows strong SIA dependency and does not transduce the CNS after systemic administration; instead, transducing the vasculature and the liver. The AAV1RX variant, which shows an intermediate SIA binding phenotype, effectively enters the brain parenchyma and transduces neurons at levels comparable to AAVrh.10. In corollary, the reciprocal swap of the AAV1RX footprint onto AAVrh.10 (AAVRX1) attenuated CNS transduction relative to AAVrh.10. We conclude that the composition of residues within the capsid variable region I (VR-I) of AAV1 and AAVrh.10 profoundly influences tropism, with altered SIA interactions playing a partial role in this phenotype. Further, we postulate a llsquo;Goldilocksrrsquo; model, wherein optimal glycan interactions can influence the CNS transduction profile of AAV capsids.

    IMPORTANCE Understanding how viruses cross the blood-brain barrier can provide insight into new approaches to block infection by pathogens or ability to exploit these pathways for designing new recombinant viral vectors for gene therapy. In this regard, modulation of virus-carbohydrate interactions by mutating the virion shell can influence the ability of recombinant viruses to cross the vascular barrier, enter the brain and enable efficient gene transfer to neurons.

  • Antibody-induced internalization of HIV-1 Env proteins limits the surface expression of the closed conformation of Env [Virus-Cell Interactions]

  • To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of ADCC. Using flow cytometry and confocal microscopy we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibility of infected cells to antibody-dependant cellular cytotoxicity (ADCC). Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells.

    IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus a better understanding this mechanism might help develop antibodies with improved capacities to mediate ADCC.

  • Antarctic penguins as reservoirs of diversity for avian avulaviruses [Genetic Diversity and Evolution]

  • Wild birds harbour a huge diversity of avian avulaviruses (formerly avian paramyxoviruses). Antarctic penguin species have been screened for avian avulaviruses since the 1980s, and as such are known hosts of these viruses. In this study we screened three penguin species from the South Shetland Islands and the Antarctic Peninsula for avian avulaviruses. We show that Adelie Penguins (Pygoscelis adeliae) are hosts for four different avian avulavirus species: the recently described avian avulaviruses 17-19, and avian avulavirus 10-like, never before isolated in Antarctica. A total of 24 viruses were isolated and sequenced; avian avulavirus 17 was the most common, and phylogenetic analysis demonstrated patterns of occurrence with different genetic clusters corresponding to penguin age and location. Following infection in specific pathogen free chickens, all four avian avulavirus species were shed from the oral cavity for up to seven days post-infection. There was limited shedding from the cloaca in a proportion of infected chickens, and all but one bird seroconverted by day 21. No clinical signs were observed. Taken together, we propose that penguin species, including Antarctic penguins, may be the central reservoir for a diversity of avian avulavirus species, and that these viruses have the potential to infect other avian hosts.

    IMPORTANCE Approximately 99% of all viruses are still to be described, and in our changing world any one of these unknown viruses could potentially expand their host range and cause epidemic disease in wildlife, agricultural animals or humans. Avian avulavirus 1 causes outbreaks in wild birds and poultry and is thus well described. However, for many avulavirus species, only a single specimen has been described, and viral ecology and epidemiology is unknown. Through the detection of avian avulaviruses in penguins from Antarctica, we have been able to expand upon our understanding of three avian avulavirus species (avian avulaviruses 17-19) and report a potentially novel avulavirus species. Importantly we show that penguins appear to play a key role in the epidemiology of avian avulaviruses and encourage additional sampling of this avian group.

  • HSV-2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation [Pathogenesis and Immunity]

  • Herpes Simplex Virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread amongst the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital RT-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG, but also the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection, and that these reactivations likely contribute to viral pathogenesis.

    IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

  • Two Co-Selected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Non-nucleoside RT Inhibitors and Nucleoside Analogs [Vaccines and Antiviral Agents]

  • Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel non-nucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the "primer grip" region and could be influenced by the M230I mutation.

    Importance: Although anti-retroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

  • Conformational engineering of HIV-1 Env based on mutational tolerance in the CD4 and PG16 bound states [Structure and Assembly]

  • HIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a llsquo;closedrrsquo; to llsquo;openrrsquo; conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex, and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface, and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.

    IMPORTANCE HIV-1 Env is dynamic, and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, amongst other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between subdomains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

  • Cell entry-independent role for the reovirus {mu}1 protein in regulating necroptosis and the accumulation of viral gene products [Virus-Cell Interactions]

  • The reovirus outer capsid protein mmu;1 regulates cell death in infected cells. To distinguish between the role of incoming, capsid-associated and newly synthesized mmu;1, we used siRNA-mediated knockdown. Loss of newly synthesized mmu;1 protein does not impact apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of mmu;1 also impacts aspects of viral replication. We found that while mmu;1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus strand RNA, plus strand RNA, and proteins that are not targeted by the mmu;1 siRNA accumulate to a greater extent when compared to control siRNA-treated cells. Furthermore, we observe a decrease in sensitivity of these viral products to inhibition by GuHCl (which targets minus strand synthesis to produce dsRNA) when mmu;1 is knocked down. Following mmu;1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of mmu;1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to a mmu;1 knockdown potentiates RIP3 dependent cell death.

    IMPORTANCE We use mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for reovirus outer capsid protein mmu;1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection. mmu;1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

  • A Novel Murine Model Expressing a Chimeric mSCARB2/hSCARB2 Receptor is Highly Susceptible to Oral Infection with Clinical Isolates of EV71 [Pathogenesis and Immunity]

  • EV71 infection is generally associated with hand-foot-mouth disease (HFMD), and may cause severe neurological disorders and even death. An effective murine oral infection model for studying the pathogenesis of various clinical EV71 isolates is lacking. We developed a transgenic mouse that expresses EV71 receptor, that is human scavenger receptor class B member 2 (hSCARB2), in a pattern highly similar to that of murine endogenous mSCARB2 protein. A FLAG-tagged SCARB2 cDNA fragment composed of exon 3-12 was inserted in murine Scarb2 gene-containing bacterial artificial chromosome (BAC) clone, and the resulting transgene was used for establishment of chimeric receptor-expressing transgenic (Tg) mice. The Tg mice intragastrically (i. g.) infected with the clinical isolates of EV71 showed neurological symptoms, such as ataxia and paralysis, and fatality. There was an age-dependent decrease in the susceptibility to viral infection. Pathological characteristics of the infected Tg mice resembled those of encephalomyelitis in human patients. Viral infection was accompanied by microglial activation. Clodronate treatment of the brain slices from Tg mice enhanced viral replication, while lipopolysaccharide treatment significantly inhibited it, suggesting an antiviral role for microglia during EV71 infection. Taken together, this Tg mouse provides a model that closely mimics the natural infection for studying EV71 pathogenesis and for evaluating the efficacy of vaccine or other antiviral drugs.

    IMPORTANCE Availability of a murine model of EV71 infection is beneficial to the understanding of pathogenic mechanism and to development and assessment of vaccine and antiviral drugs. However, the lack of a murine oral infection model thwarted the study of pathogenesis induced by the clinically relevant EV71 strain that is transmitted via the oral-oral or oral-fecal routes. Our Tg mice could be intragastrically infected with the clinically relevant EV71 strains in an efficient way, and developed neurological symptoms and pathologic changes strikingly resembling those of human infection. Moreover, these mice showed an age-dependent change in susceptibility that is similar to the human case. In prospect, this Tg mouse, when combined with the uses of other genetically modified mice, potentially contributes to studying the relationship between developmental changes in immunity and the susceptibility to virus.

  • Mutations that increase the stability of the post-fusion gp41 conformation of the HIV-1 envelope glycoprotein are selected by both an X4 and R5 HIV-1 virus to escape fusion inhibitors corresponding to heptad repeat 1 of gp41, but the gp120 adaptive mutations differ between the two viruses [Vaccines and Antiviral Agents]

  • Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) co-receptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad-repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either the HR1 or second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition, but different gp120-gp41 interactions to regulate Env conformational changes.

    IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of the envelope glycoprotein (Env) refolds into a six-helix bundle (6HB) after binding to cellular receptors. Peptides corresponding to the first or second gp41 heptad repeats (HR1 or HR2, respectively) interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors, but only the key HR1 mutation to escape a trimer-stabilized, HR1 peptide inhibitor. Here we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide, but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.

  • The N-terminus of the human cytomegalovirus glycoprotein O is important for binding to the cellular receptor PDGFR{alpha} [Virus-Cell Interactions]

  • The HCMV glycoprotein complex gH/gL/gO is required for the infection of cells by cell-free virions. It was recently shown that entry into fibroblasts depends on the interaction of gO with the platelet-derived growth factor receptor alpha (PDGFR-alpha). This interaction can be blocked with soluble PDGFR-alpha-Fc, which binds to HCMV virions and inhibits entry. The aim of this study was to identify parts of gO that contribute to PDGFR-alpha binding. In a systematic mutational approach, we targeted potential interaction sites by exchanging conserved clusters of charged amino acids of gO with alanines. To screen for impaired interaction with PDGFR-alpha, virus mutants were tested for sensitivity to inhibition by soluble PDGFR-alpha-Fc. Two mutations within the N-terminus of gO (aa56-61 and aa117-121) were partially resistant to neutralization. To validate whether these mutations impair interaction with PDGFR-alpha-Fc, we compared binding of PDGFR-alpha-Fc to mutant and wild type virions via quantitative immunofluorescence analysis. PDGFR-alpha-Fc staining intensities were reduced by 30-60% with mutant virus particles as compared to wild type particles. In concordance with the reduced binding to the soluble receptor, virus penetration into fibroblasts, which relies on binding to the cellular PDGFR-alpha, was also reduced. In contrast, PDGFR-alpha-independent penetration into endothelial cells was unaltered, demonstrating that the phenotypes of the gO mutant viruses were specific for the interaction with PDGFR-alpha. In conclusion, the mutational screening of gO revealed that the N-terminus of gO contributes to efficient spread in fibroblasts by promoting the interaction of virions with its cellular receptor.

    Importance The human cytomegalovirus is a highly prevalent pathogen that can cause severe disease in immunocompromised hosts. Currently used drugs successfully target the viral replication within the host cell, but their use is restricted due to side-effects and the development of resistance. An alternative approach is the inhibition of virus entry for which understanding the details of the initial virus-cell interaction is desirable. As binding of the viral gH/gL/gO complex to the cellular PDGFR-alpha drives infection of fibroblasts, this is a potential target for inhibition of infection. Our mutational mapping approach suggests the N-terminus as the receptor binding portion of the protein. The respective mutants were partially resistant to inhibition by PDGFR-alpha-Fc but also attenuated for infection of fibroblasts, indicating that such mutations have little if any benefit for the virus. These findings highlight the potential of targeting the interaction of gH/gL/gO with PDGFR-alpha for therapeutic inhibition of HCMV.

  • Foot-and-mouth disease virus antagonizes NOD2-mediated antiviral effects by inhibition of NOD2 protein expression [Cellular Response to Infection]

  • The role of nucleotide-binding oligomerization domain 2 (NOD2) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that FMDV infection activated NOD2-mediated IFN-bbeta; and NF-ĸB signaling pathways. NOD2 inhibited FMDV replication in the infected cells. FMDV infection triggered NOD2 transcription, while it reduced the abundance of NOD2 protein. Our results revealed that FMDV 2B, 2C, and 3C proteinase (3Cpro) were responsible for the decrease in NOD2 protein levels. 3Cpro is a viral proteinase that can cleave multiple host proteins and limit protein synthesis. Our previous studies determined that FMDV 2B suppressed protein expression of RIG-I and LGP2. Here, we found that 3Cpro and 2B also decreased NOD2 expression. However, this is the first report that 2C-induced the reduction of NOD2 protein levels. We determined that both 2B- and 2C-induced decreases in NOD2 were independent of the cleavage of host eukaryotic translation initiation factor 4 gamma (eIF4G), induction of cellular apoptosis, or proteasome, lysosome, and caspase pathways. The interactions between NOD2 and 2B or 2C were observed in the context of viral infection. The carboxyl terminal amino acids 105nndash;114 and 135nndash;144 of 2B were essential for the reduction of NOD2, while the residues 105nndash;114 were required for the interaction. Amino acids 116nndash;260 of the carboxyl terminus of 2C were essential for the interaction, while truncated 2C mutants did not reduce NOD2. These data suggested novel antagonistic mechanisms of FMDV that were mediated by 2B, 2C, and 3Cpro proteins.

    IMPORTANCE NOD2 was identified as a cytoplasmic viral pattern-recognition receptor in 2009. Subsequently, many viruses were reported to activate NOD2-mediated signaling pathways. This study demonstrated that FMDV infection activated NOD2-mediated IFN-bbeta; and NF-ĸB signaling pathways. Host cells have developed multiple strategies against viral infection; however, viruses have evolved many strategies to escape host defenses. FMDV has evolved multiple mechanisms to inhibit host type I IFN production. Here, we showed that NOD2 suppressed FMDV replication during viral infection. FMDV 2B, 2C, and 3Cpro decreased NOD2 protein expression by different mechanisms to promote viral replication. This study provided new insight into the immune evasion mechanisms mediated by FMDV and identified 2B, 2C, and 3Cpro as antagonistic factors for FMDV to evade host antiviral responses.

  • An immunodominant and conserved B-cell epitope in the envelope of simian foamy virus recognized by humans infected with zoonotic strains from apes [Pathogenesis and Immunity]

  • Cross-species transmission of simian foamy viruses (SFVs) from nonhuman primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently nonpathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (PFV, SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected, and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N96-V110 located in the leader peptide, gp18LP. Forty-three variant peptides with truncations, alanine substitutions, or amino-acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old-World monkey SFV species. The magnitude of binding to peptide N96-V110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodominant B-cell epitope recognized by humans following zoonotic SFV infection.

    Importance Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. SFVs can be transmitted to humans, in whom they establish persistent infection, like the simian lenti- and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). This is the first identification of a SFV-specific B-cell epitope recognized by human plasma samples. The immunodominant epitope lies in gp18LP, probably located at the base of the envelope trimers. The NHP species the most genetically related to humans transmitted SFV strains that induced the strongest antibody responses. Importantly, this epitope is well conserved across SFV species that infect African and Asian NHPs.

  • The C-terminal end of HIV-1 viral protein U (Vpu) encodes a clade-specific determinant to antagonize BST-2 and facilitate virion release [Virus-Cell Interactions]

  • The cellular protein BST-2/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion-assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion-assembly sites. Although present in Vpus from clades B, D and G, W76 is absent in Vpus from clades A, C and H. Mutagenesis of the C-terminal region of clade C Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion-release. This impairment was even greater when the mutations were combined with mutations of Vpu's bbeta;-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion-release to less than that of virus lacking vpu. Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion-release by supporting the displacement of BST-2 from virion-assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.

    IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mis-traffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion-assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade-specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

  • Design and Characterization of Cholesterylated Peptide HIV-1/2 Fusion Inhibitors with Extremely Potent and Long-Lasting Antiviral Activity [Vaccines and Antiviral Agents]

  • HIV infection requires lifelong treatment with multiple antiretroviral drugs in a combination, which ultimately causes cumulative toxicities and drug resistance, thus necessitating the development of novel antiviral agents. We recently found that enfuvirtide (T-20)-based lipopeptides conjugated with fatty acids have dramatically increased in vitro and in vivo anti-HIV activities. Herein, a group of cholesterol-modified fusion inhibitors were characterized with significant findings. First, novel cholesterylated inhibitors, such as LP-83 and LP-86, showed the most potent activity in inhibiting divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV). Second, the cholesterylated inhibitors were highly active to inhibit T-20-resistant mutants that still conferred high resistance to the fatty acid derivatives. Third, the cholesterylated inhibitors had extremely potent activity to block HIV Env-mediated cell-cell fusion, especially a truncated minimum lipopeptide (LP-95) showing a greatly increased potency relative to its inhibition on virus infection. Fourth, the cholesterylated inhibitors could efficiently bind to both the cellular and viral membranes to exert their antiviral activities. Fifth, the cholesterylated inhibitors displayed low cytotoxicity and binding capacity with human serum albumin. Sixth, we further demonstrated that LP-83 exhibited extremely potent and long-lasting anti-HIV activity in rhesus monkeys. Taken together, the present results help our understanding on the mechanism of action of lipopeptide-based viral fusion inhibitors and facilitate the development of novel anti-HIV drugs.

    IMPORTANCE The peptide drug T-20 remains the only membrane fusion inhibitor available for treatment of viral infection, which is used in combination therapy of HIV-1 infection; however, it exhibits relatively low antiviral activity and genetic barrier to inducing resistance, calling for the continuous development for novel anti-HIV agents. In this study, we report cholesterylated fusion inhibitors showing the most potent and broad anti-HIV activities to date. The new inhibitors have been comprehensively characterized for their modes of action and druggability, including small size, low cytotoxicity, binding ability to human HSA, and especially, the extremely potent and long-lasting antiviral activity in rhesus monkeys. Therefore, the present studies have provided new drug candidates for clinical development, which can also be used as tools to probe the mechanisms of viral entry and inhibition.

  • Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions [Structure and Assembly]

  • Infection by viruses depends on a balance between capsid stability and dynamics. This study has investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability, and between thermostability and infectivity. In the FMDV capsid a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing, or in capsid assembly or stability against thermally-induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentameric interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution, N2019H, that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations, reverted both the lethal and capsid-stabilizing effects of substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentameric interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed.

    IMPORTANCE This study provides novel insights into the little known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus, and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.

  • Cellular DNA topoisomerases are required for the synthesis of hepatitis B virus covalently closed circular DNA [Genome Replication and Regulation of Viral Gene Expression]

  • In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA, and followed by removal of PFA to allow the synchronized synthesis of relaxed circular (rc) DNA and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows for systematic screening of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis, but avoiding cytotoxic effects upon long term treatment. Using this assay, we found that all the tested topoisomerase I and II poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, siRNA knockdown of topoisomerase II significantly reduced cccDNA amplification. Taken together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.

    IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We reported herein a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and discovered that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.

  • Kinetics of early innate immune activation during HIV-1 infection of humanized mice [Cellular Response to Infection]

  • Human immunodeficiency virus type-1 (HIV-1) infection is associated with aberrant immune activation, however, most model systems for HIV-1 have been used during established infection. Here, we utilize ultra-sensitive HIV-1 quantification to delineate early events during the HIV-1 eclipse, burst and chronic phases of HIV-1 infection in humanized mice. We show that very early in infection, HIV-1 suppresses peripheral type I interferon (IFN) and interferon-stimulated gene (ISG) responses, including the HIV-1 restriction factor IFI44. At the peak of innate immune activation, prior to CD4 T cell loss, HIV-1 infection differentially affects peripheral and lymphoid TLR expression profiles in T cells and macrophages. This results in a trend towards an altered activation of NFB, TBK1 and IRF3. The subsequent type I and III IFN responses result in preferential induction of peripheral ISG responses. Following this initial innate immune activation, peripheral expression of the HIV-1 restriction factor SAMHD1 returns to levels below those observed in uninfected mice, suggesting that HIV-1 interferes with their basal expression. However, peripheral cells, still retain their responsiveness to exogenous type I IFN, whereas splenic cells show a reduction in select ISG in response to IFN. This demonstrates the highly dynamic nature of very early HIV-1 infection and suggests that blocks to the induction of HIV-1 restriction factors contribute to the establishment of viral persistence.

    IMPORTANCE Human immunodeficiency virus (HIV)-1 infection is restricted to humans and some non-human primates (e.g. chimpanzee, gorilla). Alternative model systems based on SIV infection of macaques are available but do not recapitulate all aspects of HIV-1 infection and disease. Humanized mice, which contain a human immune system, can be used to study HIV-1 but only limited information on early events and immune responses are available to date. Here, we describe very early immune responses to HIV-1 and demonstrate a suppression of cell-intrinsic innate immunity. Furthermore, we show that HIV-1 infection interacts differently with innate immune responses in blood and lymphoid organs.

  • Evolution of the HIV-1 RRE during natural infection reveals nucleotide changes that correlate with altered structure and increased activity over time. [Genetic Diversity and Evolution]

  • The HIV-1 Rev Response Element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The "early" RRE was less functionally active than the "late" RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE sequence diversity varied over the course of infection with evidence of selection pressure that led to sequence convergence as disease progressed. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding.

    Importance. HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev Response Element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

  • In vivo models of oncoproteins encoded by Kaposi's sarcoma-associated herpesvirus [Gem]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus. KSHV utilizes its proteins to modify the cellular environment to promote viral replication and persistence. Some of these proteins are oncogenic, modulating cell proliferation, apoptosis, angiogenesis, genome stability, and immune responses, among other cancer hallmarks. These changes can lead to the development of KSHV-associated malignancies. In this review, we focus on animal models of KSHV oncogenic proteins that were developed to better understand KSHV tumorigenesis.

  • Sequencing framework for the sensitive detection and precise mapping of defective interfering particle-associated deletions across influenza A and B viruses. [Genetic Diversity and Evolution]

  • The mechanisms and consequences of defective interfering particle (DIP) formation during influenza virus infection remain poorly understood. The development of next generation sequencing (NGS) technologies has made it possible to identify large numbers of DIP-associated sequences, providing a powerful tool to better understand their biological relevance. However, NGS approaches pose numerous technical challenges including the precise identification and mapping of deletion junctions in the presence of frequent mutation and base-calling errors, and the potential for numerous experimental and computational artifacts. Here we detail an Illumina-based sequencing framework and bioinformatics pipeline capable of generating highly accurate and reproducible profiles of DIP-associated junction sequences. We use a combination of simulated and experimental control datasets to optimize pipeline performance and demonstrate the absence of significant artifacts. Finally, we use this optimized pipeline to reveal how the patterns of DIP-associated junction formation differ between different strains and subtypes of influenza A and B viruses and to demonstrate how this data can provide insight into mechanisms of DIP formation. Overall, this work provides a detailed roadmap for high resolution profiling and analysis of DIP-associated sequences within influenza virus populations.

    IMPORTANCE Influenza virus defective interfering particles (DIPs) that harbor internal deletions within their genomes occur naturally during infection in humans and cell culture. They have been hypothesized to influence the pathogenicity of the virus; however, their specific function remains elusive. The accurate detection of DIP-associated deletion junctions is crucial for understanding DIP biology but is complicated by an array of technical issue that can bias or confound results. Here we demonstrate a combined experimental and computational framework for detecting DIP-associated deletion junctions using next generation sequencing (NGS). We detail how to validate pipeline performance and provide the bioinformatics pipeline for groups interested in using it. Using this optimized pipeline, we detect hundreds of distinct deletion junctions generated during infection with a diverse panel of influenza viruses and use these data to test a long-standing hypothesis concerning the molecular details of DIP formation.

  • Unique Interferon Pathway Regulation by the ANDV Nucleocapsid Protein is Conferred by Phosphorylation of Serine 386 [Virus-Cell Interactions]

  • Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS) and is the only hantavirus shown to spread person-to-person and cause highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit IFNbbeta; induction may contribute to its virulence and spread. Here we analyzed IFNbbeta; regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNbbeta; signaling and that replacing ANDV residues 252-296 with a hypervariable domain (HVD) from MAPV N prevented IFNbbeta; regulation. In addition, changing ANDV residue S386 to histidine present in MAPV N, or alanine in other hantaviruses, prevented ANDV N from regulating IFNbbeta; induction. In contrast, replacing serine with phospho-serine mimetic aspartic acid (S386D) in ANDV N, robustly inhibited IRF3 phosphorylation and IFNbbeta; induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNbbeta; induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phospho-protein and pS386 as a unique determinant of IFN regulation. In this context, finding that the ANDV HVD is required for IFN regulation by S386, but dispensable for IFN regulation by D386, suggests a role for the HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV, and suggest targeting cellular kinases as potential ANDV therapeutics.

    Importance ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator, and suggest new mechanisms for hantavirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.

  • Modulation of the CCR5 receptor/ligand axis by seminal plasma and the utility of in vitro versus in vivo models [Pathogenesis and Immunity]

  • Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggests CCR5+CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 hours of SP exposure (p=0.03). Meanwhile NK cells retain the capacity to produce limited amounts of MIP-1aalpha;/MIP-1bbeta; in response to anti-HIV-1 antibody-dependent stimulation (median 9.5% MIP-1aalpha;+MIP-1bbeta;+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three out of five animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design.

    IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

  • Essential role of enterovirus 2A protease in counteracting stress granule formation and the induction of type I interferon [Virus-Cell Interactions]

  • Most viruses have acquired mechanisms to suppress antiviral IFN-aalpha;/bbeta; and stress responses. Enteroviruses actively counteract the induction of IFN-aalpha;/bbeta; gene transcription and stress granule (SG) formation, which are increasingly implicated as a platform for antiviral signaling, but the underlying mechanisms remain poorly understood. Both viral proteases (2Apro and 3Cpro) have been implicated to suppress these responses, but these conclusions predominantly rely on ectopic overexpression of viral proteases or addition of purified viral proteases to cell lysates. Here, we present a detailed and comprehensive comparison of the effect of individual enterovirus proteases on the formation of SGs and the induction of IFN-aalpha;/bbeta; gene expression in infected cells, for representative members of the enterovirus species EV-A to EV-D. First, we show that SG formation and IFN-bbeta; induction are suppressed in cells infected with EV-A71, CV-B3, CV-A21 and EV-D68. By introducing genes encoding CV-B3 proteases in a recombinant encephalomyocarditis virus (EMCV) that was designed to efficiently activate antiviral responses, we show that CV-B3 2Apro, but not 3Cpro, is the major antagonist that counters SG formation and IFN-bbeta; gene transcription, and that 2Apro's proteolytic activity is essential for both functions. 2Apro efficiently suppressed SG formation despite PKR activation and eIF2aalpha; phosphorylation, suggesting that 2Apro antagonizes SG assembly or promotes their disassembly. Finally, we show that the ability to suppress SG formation and IFN-bbeta; gene transcription is conserved in the 2Apro of EV-A71, CV-A21 and EV-D68. Collectively, our results indicate that enterovirus 2Apro plays a key role in inhibiting innate antiviral cellular responses.

    Importance Enteroviruses are important pathogens that can cause a variety of diseases in humans, including aseptic meningitis, myocarditis, hand-foot-and-mouth disease, conjunctivitis and acute flaccid paralysis. Like many other viruses, enteroviruses must counteract antiviral cellular responses to establish an infection. It has been suggested that enterovirus proteases cleave cellular factors to perturb antiviral pathways, but the exact contribution of viral proteases 2Apro and 3Cpro remains elusive. Here, we show that 2Apro, but not 3Cpro, of all four human EV species (EV-A to EV-D) inhibits SG formation and IFN-bbeta; gene transcription. Our observations suggest that enterovirus 2Apro has a conserved function in counteracting antiviral host responses, and thereby is the main enterovirus "security protein". Understanding the molecular mechanisms of enterovirus immune evasion strategies may help to develop countermeasures to control infections with these viruses.

  • Fecal viral diversity of captive and wild Tasmanian devils characterized using virion-enriched metagenomics and meta-transcriptomics [Genetic Diversity and Evolution]

  • The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumour disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils, and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and meta-transcriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from 2 captive and 4 wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses and a gammaherpesvirus were identified, as well as known mammalian pathogens such as rabbit haemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with meta-transcriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and meta-transcriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and meta-transcriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample.

    Importance: The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumour disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (DaHV-2), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and meta-transcriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

  • Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-{kappa}B-mediated transcription [Virus-Cell Interactions]

  • Innate immunity is the first line of host defense against viral invasion. The inductions of type I interferons (IFNs) and inflammatory cytokine are essential to host antiviral immune responses, which are also key targets of viral immune evasion. Human cytomegalovirus (HCMV) can establish long-term latent infection, in which immune evasion is a pivotal step. In this study, we identified HCMV protein UL44, a DNA polymerase processivity factor, as an inhibitor of IRF3- and NF-B-dependent antiviral response. Ectopic expression of UL44 inhibited HCMV-triggered induction of downstream effector genes and enhanced viral replication. Conversely, knockdown of UL44 potentiated HCMV-triggered induction of downstream antiviral genes. UL44 interacted with IRF3 and p65, and inhibited binding of IRF3 and NF-B to the promoters of their downstream antiviral genes. These findings reveal an important mechanism of immune evasion by HCMV at the transcriptional level.

    IMPORTANCE Inductions of Type I IFNs and inflammatory cytokines play pivotal roles in host antiviral innate immune responses. Viruses have evolved various mechanisms to interfere with these processes. HCMV causes severe ailments in immunodeficient populations and is a major cause of birth defects. It has been shown that HCMV antagonizes host innate immune defenses, which is important for establishing immune evasion and latent infection. In this study, we identified the HCMV DNA polymerase subunit UL44 as a suppressor of antiviral innate immune responses. Overexpression of UL44 impaired HCMV-triggered induction of type I IFNs and other antiviral genes thus potentiated viral replication, whereas UL44-deficiency showed opposite effects. Mechanistic studies indicated that UL44 acts by inhibiting the binding of IRF3 and NF-B to the promoters of downstream antiviral genes. These findings defined an important mechanism of HCMV immune evasion at the transcriptional level, which may provide a therapeutic target for the treatment of HCMV infection.

  • Cell-to-cell spread-blocking activity is extremely limited in the sera of HSV-1 and HSV-2 infected subjects [Pathogenesis and Immunity]

  • Herpes Simplex Virus (HSV) 1 and 2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating: (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity and (iii) their capability to inhibit the cell-to-cell virus passage in vitro. All of the sera were capable of binding the gD, the gB and whole virions and all sera significantly neutralized cell-free virus. However, neither whole sera, nor purified serum IgG fraction were able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.

    Importance: Despite its importance in the physiopathology of Herpes Simplex Virus type 1 and 2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggests that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be under represented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

  • The Transcriptome of Latent Human Cytomegalovirus [Gem]

  • The latent human cytomegalovirus (HCMV) transcriptome has been extremely difficult to define due to the scarcity of naturally latent cells and the complexity of available models. The genomic era offers many approaches of transcriptome profiling that hold great potential for elucidating this challenging issue. The results from two recent studies applying different transcriptomic methodologies and analyses on both experimental and natural samples challenge the dogma of a restricted latency-associated transcription program. Instead they portray that the hallmark of HCMV latent infection is low level expression of a broad spectrum of the canonical viral lytic genes.

  • A novel fluorescent and bioluminescent Bi-Reporter influenza A virus (BIRFLU) to evaluate viral infections [Genome Replication and Regulation of Viral Gene Expression]

  • Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select fluorescent or luciferase, depending on the type of study. To circumvent this limitation, we have engineered a novel recombinant replication-competent bi-reporter IAV (BIRFLU), expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed comparable growth kinetics to wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows to monitor viral inhibition by fluorescence or bioluminescence, overcoming limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections.

    IMPORTANCE The influenza A virus (IAV) causes human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable reporter fluorescent or luciferase protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we have generated, for the first time, a recombinant replication-competent bi-reporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections in vitro and in vivo. The combination of cutting edge techniques from molecular biology, animal research and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection.

  • Contribution of the cytoplasmic determinants of Vpu to the expansion of virus containing compartments in HIV-1 infected macrophages [Virus-Cell Interactions]

  • HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+ T cells or model cell lines but the interplay between Vpu and BST2 in VCCs formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts on the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of Vpu transmembrane domain, the dileucine-like motif E59XXXLV64 and the phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.

    IMPORTANCE HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable to prevent the specific accumulation of BST2 in these structures.

  • The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH to Protect Spike Protein and Promote Release of Infectious Virus [Virus-Cell Interactions]

  • Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane when compared IBV WT infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.

    IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or anti-viral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite to potential vaccines as well as in identifying novel antiviral therapeutics.

  • ZAP, a CCCH-Type Zinc Finger Protein inhibits PRRSV replication and interacts with viral Nsp9 [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens affecting many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. PRRSV infection can cleave mitochondrial antiviral-signaling protein (MAVS) and inhibit the induction of type I interferon. The antiviral effector molecules that are involved in host protective responses to PRRSV infection are not fully understood. Here, by using transcriptome sequencing, we report that a zinc finger antiviral protein, ZAP, is up-regulated in MAVS transfected Marc-145 cells, and that ZAP suppresses PRRSV infection at the early stage of replication. We also found that the viral protein Nsp9, a RNA-dependent RNA polymerase (RdRp) interacts with ZAP. The interacting locations were mapped to the zinc finger domain of ZAP and the N-terminal amino acids 150-160 of Nsp9. These findings suggest that ZAP is an effective antiviral factor for suppressing PRRSV infection, and it sheds light on virus-host interaction.

    IMPORTANCE PRRSV continues to adversely impact the global swine industry. It is important to understand the various antiviral factors against PRRSV infection. Here, a zinc finger protein, termed ZAP, was screened from MAVS induced antiviral genes by transcriptome sequencing, and it remarkably suppresses PRRSV replication and interacts with PRRSV Nsp9. The zinc finger domain of ZAP and the 150-160 amino acids of Nsp9 are responsible for the interaction. These findings expand the antiviral spectrum of ZAP, and provide a better understanding of ZAP antiviral mechanisms, as well as virus-host interactions.

  • Humanization of the murine neutralizing antibodies against human herpesvirus 6B [Vaccines and Antiviral Agents]

  • Exanthem subitum is a common childhood illness caused by Human herpesvirus 6B (HHV-6B) primary infection. It is occasionally complicated by febrile seizure and even encephalitis. The HHV-6B reactivation also causes encephalitis especially after allogeneic hematopoietic stem cell transplantation. However, no adequate antiviral treatment for HHV-6B has yet been established. Mouse-derived monoclonal antibodies (Mabs) against the envelope glycoprotein complex gH/gL/gQ1/gQ2 of HHV-6B have been shown to neutralize the viral infection. These antibodies have the potential to become antiviral agents against HHV-6B despite their inherent immunogenicity to the human immune system. Humanization of Mabs derived from other species is one of the proven solutions to such a dilemma. Here, we constructed chimeric forms of two neutralizing Mabs against HHV-6B to make humanized antibodies. Both showed neutralizing activities equivalent to those in their original ones. This is the first report of humanized antibodies against HHV-6B and provides a basis for the further development of HHV-6B-specific antivirals.

    Importance

    Human herpesvirus 6B (HHV-6B) establishes lifelong latent infection in most individuals after the primary infection. Encephalitis is the most severe complication caused by both the primary infection and the reactivation of HHV-6B, and is the considerable cause of mortality in patients without any established treatments now. Humanization of the murine neutralizing antibodies described in this research provided a feasible way to reduce the inherent immunogenicity without changing the neutralizing activities. These newly designed chimeric antibodies against HHV-6B have the potential to be candidates of antiviral for future use.

  • SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1 [Virus-Cell Interactions]

  • SUMOylation is a post-translational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. Here, we found that the VP1 protein, the RNA dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that the residues 404I406I within SUMO interaction motif 3 of VP1 is the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. Reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.

    IMPORTANCE

    SUMOylation is an extensively discussed post-translational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation was residues 404I406I of its SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.

  • Development of an ORF45-derived peptide to inhibit the sustained RSK activation and lytic replication of Kaposi's sarcoma-associated herpesvirus [Vaccines and Antiviral Agents]

  • The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) requires sustained ERK-RSK activation, which is induced by an immediately early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease of late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56-76 aa region and HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis.

    Importance

    ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decrease lytic replication, indicating that the ORF45-RSK association is unique target for KSHV-related diseases. However, the side effects, low affinity and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia- or chemical-induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

  • Bridging vaccine-induced HIV-1 neutralizing and effector antibody responses in rabbit and rhesus macaque animal models [Vaccines and Antiviral Agents]

  • Studies in animal models are essential prerequisites for clinical trials of candidate HIV vaccines. Small animals, such as rabbits, are used to evaluate promising strategies prior to further immunogenicity and efficacy testing in non-human primates. Our goal was to determine how HIV-specific vaccine-elicited antibody responses, epitope specificity, and Fc-mediated functions in the rabbit model can predict those in the rhesus macaque (RM) model. Detailed comparisons of the HIV-1 specific IgG response were performed on serum from rabbits and RM given identical MVA-prime/gp120-boost immunization regimens. We found that vaccine-induced neutralizing antibody, gp120-binding antibody levels and immunodominant specificities, antibody dependent cellular phagocytosis of HIV-1 virions, and antibody-dependent cellular cytotoxicity (ADCC) responses against gp120-coated target cells were similar in rabbits and RM. However, we also identified characteristics of humoral immunity that differed across species. ADCC against HIV-infected target cells was elicited in rabbits but not in RM and we observed differences among subdominantly targeted epitopes. Human Fc-receptor binding assays and analysis of antibody-cell interactions indicated that rabbit vaccine-induced antibodies effectively recruited and activated human natural killer cells, while vaccine-elicited RM antibodies were unable to activate either human or RM NK cells. Thus, our data demonstrates that both Fc-independent and Fc-dependent functions of rabbit antibodies can be measured with commonly used in vitro assays, however the ability of immunogenicity studies performed in rabbits to predict responses in RM will vary depending on the particular immune parameter of interest.

    IMPORTANCE

    Non-neutralizing antibody functions have been associated with reduced infection risk, or control of virus replication, for HIV-1 and related viruses. It is therefore critical to evaluate development of these responses throughout all stages of preclinical testing. Rabbits are conventionally used to evaluate the ability of vaccine candidates to safely elicit antibodies that bind and neutralize HIV-1. Yet, it remained unexplored how effectively rabbits model the development of non-neutralizing antibody responses in primates. We administered identical HIV-1 vaccine regimens to rabbits and rhesus macaques and performed detailed comparisons of vaccine-induced antibody responses. We demonstrated that non-neutralizing HIV-specific antibody responses can be studied in the rabbit model, and have identified aspects of these responses that are common, and those that are unique, to rabbits and rhesus macaques. Our findings will help determine how to best utilize preclinical rabbit and rhesus macaque models to accelerate HIV vaccine candidate testing in human trials.

  • A humanized mouse model of HIV-1 latency with enrichment of latent virus in PD-1+ and TIGIT+ CD4 T cells [Pathogenesis and Immunity]

  • Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semi-quantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently-infected human CD4 T cells can be readily detected in mouse lymphoid tissues, and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. Additionally, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently-infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.

    Importance: HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses, and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART, and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

  • Divergent HIV-1 Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques [Vaccines and Antiviral Agents]

  • Most HIV-1 infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (IM) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by intranasal (IN) and intramuscular (IM) routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single IM immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the IM route had high peripheral T follicular helper (pTfh) cells in blood, but low Tfh in lymph nodes. Conversely, animals immunized by the IN route had high Tfh in lymph nodes, but low pTfh in the blood. Animals immunized by only the IM route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal IN route had higher ADCC antibodies. When these Env immunized animals were challenged rectally with SHIVSF162P3, they all became infected. However, mucosally-SC-Ad immunized animals had lower viral loads their gastrointestinal tracts. These data suggest there may be benefits in educating the immune system at mucosal sites during HIV vaccination.

    Importance

    HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle make a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

  • Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus Is a New Inhibitor of Hepatitis C Virus RNA Replication [Vaccines and Antiviral Agents]

  • Recent emergence of direct acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, search for new inhibitors and with lower cost of production should be pursued. In this context, crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed isolating and identifying the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (EC50 = 1.35 mmu;M). This antiviral activity was confirmed with a HCV subgenomic replicon and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir resistant mutants (L31M or Y93H in NS5A). Finally, resistant mutants to dehydrojuncusol were obtained and showed that HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting NS5A protein and is active against HCV resistant variants frequently found in patients with treatment failure.

    Importance

    Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of the treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to appearance of viral resistance. Here we report on the identification of a new HCV inhibitor, dehydrojuncusol that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species very common in the coastlines worldwide. This molecule might serve as a lead for the development of new therapy more accessible to hepatitis C patients in the future.

  • A lysine residue essential for geminivirus replication also controls nuclear localization of the TYLCV Rep protein. [Virus-Cell Interactions]

  • Geminiviruses are ssDNA viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato Yellow Leaf Curl Virus (TYLCV) with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato Golden Mosaic Virus (TGMV) to interact with the E2 SUMO conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1-binding differs depending on the Rep protein examined.

    Importance

    Nuclear activity of the Replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization is mapped for any geminiviral Rep protein. Our data adds another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2 conjugating enzyme.

  • CD4- and time-dependent susceptibility of HIV-1-infected cells to ADCC [Pathogenesis and Immunity]

  • HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1+ individuals predominantly target CD4-induced (CD4i) epitopes on HIV-1 envelope glycoprotein (Env). These CD4i epitopes are usually concealed on the surface of infected cells due to CD4-downregulation by the HIV-1 accessory proteins Nef and Vpu. We hypothesized that early-stage infected cells in the process of downregulating CD4 could be more susceptible to ADCC compared to late-stage infected cells that have fully downregulated CD4. There was significantly higher binding of antibodies within plasma from HIV-1-infected individuals to early-stage CD4-intermediate infected cells compared to late-stage CD4-low infected cells. However, we noted HIV-1-uninfected bystander cells and HIV-1-infected cells, at various stages of downregulating CD4, were all susceptible to NK cell-mediated ADCC. Importantly, we observed that the cytolysis of bystander cells and early infected cells in this culture system was driven by sensitization of target cells by inoculum-derived HIV-1 Env or virions. This phenomenon provided Env to target cells prior to de novo Env expression, resulting in artefactual ADCC measurements. Future studies should take into consideration the inherent caveats of in vitro infection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection.

    IMPORTANCE

    An increasing body of evidence suggests that ADCC contributes to protection against HIV-1 acquisition and slower HIV-1 disease progression. Targeting cells early during the infection cycle would be most effective in limiting virus production and spread. We hypothesized that there could be a time-dependent susceptibility of HIV-1-infected cells to ADCC in regard to CD4 expression. We observed NK cell-mediated ADCC of HIV-1-infected cells at multiple stages of CD4 downregulation. Importantly, ADCC of early infected cells appeared to be driven by a previously unappreciated problem of soluble Env and virions from the viral inoculum sensitizing uninfected cells to ADCC prior to de novo Env expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

  • Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The papillomavirus (PV) E2 protein coordinates viral transcription and genome replication. Following a strategy to identify amino acids in E2 that are post-translationally modified, we reported that tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) complexed with and phosphorylated E2, which inhibited viral DNA replication. Here we present several lines of evidence indicating that tyrosine (Y) 138 of HPV-31 E2 is a substrate of FGFR3. The active form of FGFR3 bound to and phosphorylated the region of amino acids 107-175 in HPV-31 E2. The E2 phenylalanine (F) mutant Y138F displayed reduced FGFR3-induced phosphotyrosine. A constitutive kinase active FGFR3 inhibited WT E2 induced E1-dependent DNA replication while the 138F mutant retained activity. The tyrosine to glutamic acid (E) mutant Y138E, which can mimic phosphotyrosine, failed to induce transient DNA replication although it maintained ability to bind and localize the viral DNA helicase E1 to the viral origin. The bromodomain-containing protein 4 (Brd4) binds to E2 and is necessary for initiation of viral DNA synthesis. Interestingly, the Y138E protein co-immunoprecipitated with full length Brd4 but was defective for association with its C-terminal domain (CTD). These results imply that the activity of the FGFR3 kinase in the infected epithelial cell restricts the HPV replication program through phosphorylation of E2 at Y138, which interferes with E2 binding to the Brd4 CTD, and that this interaction is required for initiation of viral DNA synthesis.

    IMPORTANCE Human papillomaviruses (HPV) are highly infectious pathogens that commonly infect the oropharynx and uterine cervix. The idea that post-translational modifications of viral proteins coordinate viral genome replication is less explored. We recently discovered that fibroblast growth factor receptor 3 phosphorylates the viral E2 protein. The current study demonstrates that FGFR3 phosphorylates E2 at tyrosine 138, which inhibits association with the C-terminal peptide of Brd4. This study illustrates a novel regulatory mechanism of virus-host interaction and provides insight into the role of Brd4 in viral replication.

  • MC159 of molluscum contagiosum virus suppresses autophagy by recruiting cellular SH3BP4 via an SH3 domain-mediated interaction [Virus-Cell Interactions]

  • MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-B-mediated transcription and suppression of pro-apoptotic caspase activation, but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159, and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.

    IMPORTANCE

    After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large especially in immunocompromised individuals. While not life-threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.

  • Biphasic functional interaction between the adenovirus E4orf4 protein and DNA-PK [Virus-Cell Interactions]

  • The adenovirus (Ad) E4orf4 protein contributes to viral-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by expression of E4orf4 alone. In this report we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with DNA-PK and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection, but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation, as well as by co-localization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell-death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

    Importance

    Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR) which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

  • Evidence for both intermittent and persistent compartmentalization of HIV-1 in the female genital tract [Genetic Diversity and Evolution]

  • HIV-1 has been shown to evolve independently in different anatomical compartments but studies in the female genital tract have been inconclusive. Here we examined evidence of compartmentalization using HIV-1 subtype C envelope (Env) glycoprotein genes (gp160) obtained from matched cervico-vaginal lavage (CVL) and plasma samples over 2-3 years of infection. HIV-1 gp160 amplification from CVL was achieved for only four of 18 acutely infected women and this was associated with the presence of pro-inflammatory cytokines and/or measurable viremia in the CVL. Maximum likelihood trees and divergence analyses showed that all four individuals had monophyletic compartment-specific clusters of CVL and/or plasma-derived gp160 sequences at all or some timepoints. However, two participants (CAP177 and CAP217) had CVL gp160 diversity patterns that differed from those in plasma and showed restricted viral flow from the CVL. Statistical tests of compartmentalization revealed evidence of persistent compartment-specific gp160 evolution in CAP177, while in CAP217 this was intermittent. Lastly, we identified several Env sites that distinguished viruses in these two compartments; for CAP177, amino acid differences arose largely through positive selection while indels were more common in CAP217. In both cases these differences contributed to substantial charge changes spread across the Env. Our data indicates that, in some women, HIV-1 populations within the genital tract can have Env genetic features that differ from those of viruses in plasma: a factor which could potentially impact the sensitivity of viruses in the genital tract to vaginal microbicides and vaccine-elicited antibodies.

    IMPORTANCE

    Most HIV-1 infections in sub-Saharan Africa are acquired heterosexually through the genital mucosa. Understanding the properties of viruses replicating in the female genital tract, and whether these properties differ from those of more commonly studied viruses replicating in the blood, is therefore important. Using longitudinal CVL and plasma-derived sequences from four HIV-1 subtype C infected women we found fewer viral migrations from the genital tract to plasma than in the opposite direction, suggesting a mucosal sieve effect from the genital tract to the blood compartment. Evidence for both persistent and intermittent compartmentalization between the genital tract and plasma viruses during chronic infection was detected in two of four individuals; perhaps explaining previously conflicting findings. In cases where compartmentalization occurred, comparison of CVL and plasma-derived HIV sequences indicated that distinct features of viral populations in the CVL may impact the efficacy of microbicides and vaccines designed to provide mucosal immunity.

  • Quantitative proteomic analysis reveals unfolded protein response involved in severe fever with thrombocytopenia syndrome virus infection [Cellular Response to Infection]

  • Severe fever with thrombocytopenia syndrome (SFTS) is an emerging, highly pathogenic, infectious disease caused by infection with a newly discovered tick-borne phlebovirus, SFTS virus (SFTSV). Limited information on the molecular mechanism of SFTSV infection and pathogenesis impedes the development of effective vaccines and drugs for SFTS prevention and treatment. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis of SFTSV-infected HEK 293 cells was performed to explore dynamic host cellular protein responses towards SFTSV infection. A total of 433 out of 5,606 host proteins involved in different biological processes were differentially regulated by SFTSV infection. The proteomic results highlighted a potential role of endoplasmic reticular stress-triggered unfolded protein response (UPR) in SFTSV infection. Further functional studies confirmed that all three major branches of the UPR, including the PRKR-like endoplasmic reticulum kinase (PERK), the activating transcription factor-6 (ATF6) and the inositol-requiring protein-1 (IRE1)-X-box-binding protein 1 (XBP1) pathways, were activated by SFTSV. However, only the former two pathways play a crucial role in SFTSV infection. Furthermore, expression of SFTSV glycoprotein (GP) alone was sufficient to stimulate the UPR, while suppression of PERK and ATF6 notably decreased GP expression. Interestingly, two other newly discovered phleboviruses, Heartland virus (HRTV) and Guertu virus (GTV), also stimulated the UPR, suggesting a common mechanism shared by these genetically related phleboviruses. This study provides a global view to our knowledge on how host cells respond to SFTSV infection and highlights that host cell UPR plays an important role in phlebovirus infection.

    IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe fever with thrombocytopenia syndrome in humans, with a mortality rate reaching up to 30% in some outbreaks. There are currently no FDA-approved vaccines or specific antivirals available against SFTSV. To comprehensively understand the molecular interactions occurring between SFTSV and the host cell, we exploit quantitative proteomic approach to investigate the dynamic host cellular responses to SFTSV infection. The results highlight multiple biological processes being regulated by SFTSV infection. Among these, we focused on exploration of the mechanism of how SFTSV infection stimulates the host cell's unfolded protein response (UPR) and identified the UPR as a common feature shared by SFTSV-related new emerging phleboviruses. This study, for the first time to our knowledge, provides a global map for host cellular responses to SFTSV infection and highlighted potential host targets for further research.

  • Adenovirus 5 E1A interacts with E4orf3 to regulate viral chromatin organization [Genome Replication and Regulation of Viral Gene Expression]

  • Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units that each express a variety of proteins. Chief amongst these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N-terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, that is restrictive to viral gene expression and that E1A overcomes this via a direct interaction with E4orf3. We also show that during infection E1A co-localizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. Inability of E1A to interact with E4orf3 has significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlights a novel mechanism that regulates viral gene expression.

    IMPORTANCE

    To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

  • A highly unusual V1 region of Env in an elite controller of HIV infection [Pathogenesis and Immunity]

  • HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of anti-retroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19 years, the last 10 of which were in the absence of anti-retroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.

    Importance: Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well-known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities.

  • Guinea Fowl Coronavirus Diversity has Phenotypic Consequences for Glycan and Tissue Binding [Virus-Cell Interactions]

  • Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period 2014-2016, affected guinea fowl flocks were sampled in France and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can, in addition to the previously elucidated biantennary diLacNAc receptor, bind to glycans capped with alpha 2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014-S1 has an increased affinity for these glycans compared to GfCoV/2011-S1, which was in agreement with the increased avidity of GfCoV/2014-S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before applying spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.

    IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economical losses, the detection and understanding of the molecular determinants of viral tropism is of ultimate importance. Here we set out to study those parameters and obtained in-depth insight in the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins result in differences in affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for its tissue tropism and pathogenesis in guinea fowls.

  • Species-specific pathogenicity of severe fever with thrombocytopenia syndrome virus is determined by anti-STAT2 activity of NSs [Virus-Cell Interactions]

  • Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel emerging virus that has been identified in China, South Korea, and Japan, and induces thrombocytopenia and leukocytopenia in humans with a high case fatality rate. SFTSV is pathogenic to humans, while immunocompetent adult mice and golden Syrian hamsters infected with SFTSV never show apparent symptoms. However, mice deficient for the gene encoding the aalpha; chain of the interferon (IFN) aalpha; and bbeta; receptor (Ifnar1-/- mice) and golden Syrian hamsters deficient for the gene encoding signal transducer and activator of transcription 2 (Stat2-/- hamsters) are highly susceptible to SFTSV infection, with infection resulting in death. The nonstructural protein (NSs) of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT proteins. Here, we demonstrated that SFTSV induces lethal acute disease in STAT2-deficient mice, but not in STAT1-deficient mice. Furthermore, we discovered that NSs cannot inhibit type I IFN signaling in murine cells due to an inability to bind to murine STAT2. Taken together, our results imply that the dysfunction of NSs in antagonizing murine STAT2 can lead to inefficient replication and the loss of pathogenesis of SFTSV in mice.

    IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV), which has been reported in China, South Korea, and Japan. Here, we revealed that mice lacking STAT2, which is an important factor for antiviral innate immunity, are highly susceptible to SFTSV infection. We also show that SFTSV NSs cannot exert its anti-innate immunity activity in mice due to the inability of the protein to bind to murine STAT2. Our findings suggest that the dysfunction of SFTSV NSs as an IFN antagonist in murine cells confers a loss of pathogenicity of SFTSV in mice.

  • Interferon-{beta} contributes to astrocyte activation in the brain following reovirus infection [Virus-Cell Interactions]

  • Reovirus encephalitis in mice was used as a model system to investigate astrocyte activation (astrogliosis) following viral infection of the brain. Reovirus infection resulted in astrogliosis as evidenced by increased expression of glial fibrillary acidic protein (GFAP), and up-regulation of genes that have been previously associated with astrocyte activation. Astrocyte activation occurred in regions of the brain that are targeted by reovirus, but extended beyond areas of active infection. Astrogliosis also occurred following reovirus infection of ex vivo brain slice cultures (BSCs) demonstrating that factors intrinsic to the brain are sufficient to activate astrocytes, and that this process can occur in the absence of any contribution from the peripheral immune response. In agreement with previous reports reovirus antigen did not co-localize with GFAP in infected brains cells suggesting that reovirus does not infect astrocytes. Reovirus-infected neurons produce interferonbbeta; (IFNbbeta;). IFNbbeta; treatment of primary astrocytes resulted in both the up-regulation of GFAP and cytokines that are associated with astrocyte activation. In addition, the ability of media from reovirus-infected BSC to activate primary astrocytes was blocked by anti-IFNbbeta; antibodies. These results suggest that IFNbbeta;, likely released from reovirus-infected neurons, results in the activation of astrocytes during reovirus encephalitis. In areas where infection and injury were pronounced, an absence of GFAP staining was consistent with activation-induced cell death (AICD) as a mechanism of inflammation control. In support of this, activated Bak and cleaved caspase 3 were detected in astrocytes following reovirus infection of the mouse CNS indicating that activated astrocytes undergo apoptosis.

    IMPORTANCE Viral encephalitis is a significant cause of worldwide morbidity and mortality and specific treatments are extremely limited. Virus infection of the brain triggers neuroinflammation, however the role of neuroinflammation in the pathogenesis of viral encephalitis is unclear. Initial neuroinflammatory responses likely contribute to viral clearance but prolonged exposure to pro-inflammatory cytokines released during neuroinflammation may be deleterious and contribute to neuronal death and tissue injury. Activation of astrocytes is a hallmark of neuroinflammation. Herein we show that reovirus infection of the brain results in the activation of astrocytes via an IFNbbeta;-mediated process and that these astrocytes later die by Bak-mediated apoptosis. A better understanding of neuroinflammatory responses during viral encephalitis may facilitate the development of new treatment strategies for these diseases.

  • NK cells accumulate in infected tissues and contribute to pathogenicity of Ebola virus in mice. [Pathogenesis and Immunity]

  • Understanding the immune parameters responsible for survival following Ebola virus (EBOV) infection is paramount for developing countermeasures. In lethal EBOV infections, levels of both NK and T cells decline drastically in the circulation and lymphoid tissues before death. However, the fate of these lymphocyte in viral replication sites remains unknown. In this study, RT-PCR and FACS analysis were used to investigate lymphocyte frequencies in various mouse infected tissues after challenge with mouse-adapted EBOV (MA-EBOV). A decrease in NK cell numbers from the systemic circulation was observed concomitant to an increase of these cells in tissues that are supporting active replication of EBOV. Unexpectedly, NK accumulation in virus replication sites correlated with enhanced EBOV disease progression in specific conditions; at high challenge dose, NK depleted mice displayed lower viremia, liver damage and higher hepatic T cell level. Up regulation of UL16 binding protein 1 (ULBP-1) was detected on hepatic T cells suggesting that NK cells participate in their elimination. Overall, this study supports the concept that NK cells accumulate in EBOV infected tissues and that they can contribute to viral pathogenicity.

    Importance: Ebola virus (EBOV) outbreaks can claim numerous lives and also devastate the local health infrastructure, as well as the economy of affected countries. Lethal EBOV infection has been documented to decrease the level of several immune cells in the blood that are necessary to defend the host. This decrease in immune cells is however not observed in individuals surviving EBOV infection. Having a better grasp of how these immune cells are lost is therefore of high importance to develop and improve new and existing therapeutics. The significance of our research is in identifying the mechanism responsible for the apparent loss of immune cells in lethal EBOV infection. This will allow therapeutic options aiming at preventing the loss of these immune cells and therefore allowing infected individuals to better fight the infection.

  • HIV controllers have low inflammation associated with a strong HIV-specific immune response in blood [Pathogenesis and Immunity]

  • HIV-Controllers (HIC) maintain control of HIV replication without combined antiretroviral treatment (cART). The mechanisms leading to virus control are not fully known. We used gene expression and cellular analyses to compare HIC and HIV-1 infected individuals under cART. In the blood, HIC are characterized by a low inflammation, a down modulation of NK inhibitory cell signaling and an up regulation of T-cell activation gene expression. This balance that persists following stimulation of cells with HIV antigens, was consistent with functional analyses showing a bias towards a Th1 and cytotoxic T cell response and a lower production of inflammatory cytokines. Taking advantage of the characterization of HIC based upon their CD8+ T lymphocyte capacity to suppress HIV-infection, we show that unsupervised analysis of differentially expressed genes fits clearly with this cytotoxic activity allowing the characterization of a specific signature of HIC. These results reveal significant features of HIC making the bridge between cellular function, gene signatures and the regulation of inflammation and killing capacity of HIV-specific CD8+T cells. Moreover, these genetic profiles are consistent through analyses performed from blood to PBMC and T-cells. HIV controllers maintain strong HIV-specific immune responses with low levels of inflammation. Our findings may pave the way for new immunotherapeutic approaches leading to strong HIV-1-specific immune responses while minimizing inflammation.

    IMPORTANCE A small minority of HIV infected patients, called "HIV Controllers" (HIC) maintains spontaneous control of HIV replication. It is therefore important to identify mechanisms that contribute to the control of HIV replication that may have implications for vaccine design. We observed a low inflammation, a down modulation of natural killer inhibitory cell signaling and an up regulation of T-cell activation gene expression in blood of HIC compared to patients under combined antiretroviral treatment. This profile persists following in vitro stimulation of peripheral blood mononuclear cells with HIV antigens, and was consistent with functional analyses showing a Th1 and cytotoxic T cell response and a lower production of inflammatory cytokines. These results reveal significant features of HIV controllers that maintain strong HIV-specific immune responses with low levels of inflammation. These findings define the immune status of HIC that is probably associated with the control of viral load.

  • Role of enteroviral RNA-dependent RNA polymerase in regulation of MDA5-mediated interferon-{beta} activation [Virus-Cell Interactions]

  • Infection of enteroviruses can cause severe neurological complications in humans. The interactions between the enteroviral and host proteins may facilitate the virus replication and be involved in pathogenicity of infected individuals. It has been shown that human enteroviruses possess various mechanisms to suppress host innate immune responses in infected cells. Previous studies showed that infection of enterovirus 71 could cause degradation of MDA5, which is a critical cytoplasmic pathogen sensor in recognition of picornaviruses for initiating transcription of type I interferon. In the present study, we demonstrated that the RNA-dependent RNA polymerase (RdRP; also denoted 3Dpol) encoded by EV71 could interact with the CARD domain of MDA5 and play a role in inhibition of MDA5-mediated IFN-bbeta; promoter activation and mRNA expression. In addition, we found that the 3Dpol protein encoded by Coxsackievirus B3 could also interact with MDA5 and down-regulated the antiviral signaling initiated by MDA5. These findings indicate that enteroviral RdRP may function as an antagonist against the host antiviral innate immune response.

    IMPORTANCE Infection of enterovirus cause severe neurological complications in humans. Human enteroviruses possess various mechanisms to suppress host type I IFN response in infected cells to establish the viral replication. In the present study, we found that the enteroviral 3Dpol protein, which is viral RNA-dependent RNA polymerase for replicating viral RNA, plays a role in inhibition of MDA5-mediated IFN-bbeta; promoter activation. We further demonstrated that enteroviral 3Dpol protein interacts with the CARD domain of MDA5. These findings indicate that enteroviral RdRP functions as an antagonist against the host antiviral response.

  • LAT inhibits apoptosis via downregulation of components of type I IFN pathway during latent HSV-1 ocular infection [Pathogenesis and Immunity]

  • Herpes Simplex Virus type 1 (HSV-1) latency associated transcript (LAT) has been shown to inhibit apoptosis via inhibiting activation of pro-apoptotic caspases. However, the mechanism of LAT control of apoptosis is unclear, because LAT is not known to encode a functional protein, and the LAT transcript is found largely in the nucleus. We hypothesized that LAT inhibits apoptosis by regulating expression of genes that control apoptosis. Consequently, we sought to establish the molecular mechanism of the anti-apoptosis functions of LAT at a transcriptional level during latent HSV-1 ocular infection in mice. Our results suggest that: 1) LAT likely inhibits apoptosis via upregulation of several components of the Type I IFN pathway; 2) LAT does not inhibit apoptosis via the caspase cascade at a transcriptional level or via downregulating TLRs; 3) The mechanism of LAT antiapoptotic effect is distinct from the baculovirus cpIAP because replacement of LAT with cpIAP resulted in a different gene expression pattern than either LAT(+) or LAT(-) viruses; and 4) Replacement of LAT with cpIAP does not cause upregulation of CD8 or markers of T cell exhaustion despite having similar levels of latency, further supporting that LAT and cpIAP function via distinct mechanisms.

    IMPORTANCE HSV-1 latency-reactivation cycle is the cause of significant human pathology. HSV-1 latency associated transcript (LAT) functions by regulating latency and reactivation, in part by inhibiting apoptosis. However, the mechanism of this process is unknown. Here we show that LAT likely controls apoptosis via downregulation of several components in the JAK-STAT pathway. Furthermore, we provide evidence that immune exhaustion is not caused by the antiapoptotic activity of LAT.

  • Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures [Virus-Cell Interactions]

  • Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both aalpha;2,6-linked and aalpha;2,3-linked sialic acid (SA) receptors, which preferentially bind the HA of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropism. In accordance with distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and non-ciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected non-ciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, non-ciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37ddeg;C, however, replication of the A(H5N1) virus was significantly attenuated at 33ddeg;C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression of immune mediator genes and resulted in more cell damage/loss when compared with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.

    IMPORTANCE: Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.

  • Vaccinia virus BBK E3 ligase adaptor A55 targets importin-dependent NF-{kappa}B activation and inhibits CD8+ T-cell memory [Virus-Cell Interactions]

  • Viral infection of cells is sensed by pathogen recognition receptors that trigger an anti-viral innate immune response and consequently viruses have evolved countermeasures. Vaccinia virus (VACV) evades the host immune response by expressing scores of immunomodulatory proteins. One family of VACV proteins are the BTB-BACK domain containing, Kelch-like (BBK) family of predicted cullin-3 E3 ligase adaptors: A55, C2 and F3. Previous studies demonstrated that gene A55R encodes a protein that is non-essential for VACV replication yet affects viral virulence in vivo. Here we report that A55 is an NF-B inhibitor acting downstream of IBaalpha; degradation, preventing gene transcription and cytokine secretion in response to cytokine stimulation. A55 targets the host importin aalpha;1 (KPNA2), acting to reduce p65 binding and its nuclear translocation. Interestingly, whilst A55 was confirmed to co-precipitate with cullin-3 in a BTB-dependent manner, its NF-B inhibitory activity mapped to the Kelch domain, which alone is sufficient to co-precipitate with KPNA2 and inhibit NF-B signalling. Intradermal infection of mice with a virus lacking A55R (vA55) increased VACV-specific CD8+ T-cell proliferation, activation, and cytotoxicity in comparison to the WT virus. Furthermore, immunisation with vA55 induced increased protection to intranasal VACV challenge compared to control viruses. In summary, this report describes the first target of a poxvirus-encoded BBK protein and a novel mechanism for DNA virus immune evasion, resulting in increased CD8+ T-cell memory and a more immunogenic vaccine.

    IMPORTANCE NF-B is a critical transcription factor in the innate immune response to infection and in shaping adaptive immunity. The identification of host and virus proteins that modulate the induction of immunological memory is important for improving virus-based vaccine design and efficacy. In viruses, the expression of BTB-BACK Kelch-like (BBK) proteins is restricted to poxviruses and conserved within them, indicating the importance of these proteins for these medically important viruses. Using vaccinia virus (VACV), the smallpox vaccine, we report that the VACV BBK protein A55 dysregulates NF-B signalling by disrupting the p65-importin interaction, thus preventing NF-B translocation and blocking NF-B-dependent gene transcription. Infection with VACV lacking A55 induces increased VACV-specific CD8+ T-cell memory and better protection against VACV challenge. Studying viral immunomodulators therefore expands not only our understanding of viral pathogenesis and immune evasion strategies, but also understanding of the immune signalling cascades controlling anti-viral immunity and the development of immune memory.

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