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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating [Structure and Assembly]

  • The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an aalpha; helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33ddeg;C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33ddeg;C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33ddeg;C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33ddeg;C during the next cycle of infection.

    IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPase possessed a defect in encapsidation at 37ddeg;C and subsequently in uncoating during the next cycle of infection at 33ddeg;C. These studies not only reveal a new site in 2CATPase that is involved in encapsidation but also identify a link between encapsidation and uncoating.

  • Hepatitis B Virus Surface Antigen Activates Myeloid Dendritic Cells via a Soluble CD14-Dependent Mechanism [Cellular Response to Infection]

  • Hepatitis B virus (HBV) infection can cause chronic liver disease, which is associated with increased risk of liver cirrhosis, liver failure, and liver cancer. Clearance of HBV infection requires effective HBV-specific immunity; however, the immunological mechanisms that determine the development of effective HBV-specific immunity are poorly understood. Dendritic cells (DC) play a pivotal role in the regulation of antiviral immunity. Here, we investigated the interaction between HBV surface antigen (HBsAg), the main envelope glycoprotein of HBV, and BDCA1+ myeloid dendritic cells (mDC). Exposure of peripheral blood-derived BDCA1+ mDC to HBsAg resulted in strong DC maturation, cytokine production, and enhanced capacity to activate antigen-specific cytotoxic T cells (CTLs). By using neutralizing antibodies, crucial roles for CD14 and Toll-like receptor 4 (TLR4) in HBsAg-mediated BDCA1+ mDC maturation were identified. Concordantly, HBsAg-mediated DC maturation required fetal calf serum (FCS) or human plasma, naturally containing soluble CD14 (sCD14). Intriguingly, HBsAg-induced DC maturation was significantly reduced in umbilical cord blood plasma, which contained less sCD14 than adult plasma, indicating that sCD14 is an important host factor for recognition of HBsAg by DC and subsequent DC activation. A direct interaction between sCD14 and HBsAg was demonstrated by using enzyme-linked immunosorbent assay (ELISA). Moreover, sCD14-HBsAg complexes were detected both in vitro and in sera of HBV-infected patients. The abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1+ mDC in vivo. We conclude that HBsAg activates BDCA1+ DC via an sCD14-dependent mechanism. These findings provide important novel insights into the initiation of HBV-specific immunity and facilitate development of effective immunotherapeutic interventions for HBV.

    IMPORTANCE Hepatitis B virus (HBV) infection is a significant health problem, as it causes progressive liver injury and liver cancer in patients with chronic HBV infection, which affects approximately 250 million individuals worldwide. Some of the infected adults and the majority of neonates fail to mount an effective immune response and consequently develop chronic infection. The viral and host factors involved in the initiation of effective HBV-specific immune responses remain poorly understood. Here we identified CD14 and TLR4 as receptors for HBsAg, the main HBV envelope antigen. HBsAg induced strong maturation of dendritic cells (DC), which have a central role in regulation of virus-specific immunity. These results provide essential novel insights into the mechanisms underlying the initiation of HBV-specific immunity. Intriguingly, since neonates have naturally low sCD14, the finding that serum-derived sCD14 is a crucial host factor for recognition of HBsAg by DC may have implications for immunity of neonates to HBV infection.

  • Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters [Pathogenesis and Immunity]

  • Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1aalpha; and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS.

    IMPORTANCE Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection, but those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like in our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

  • Monoclonal Antibodies to Different Components of the Human Cytomegalovirus (HCMV) Pentamer gH/gL/pUL128L and Trimer gH/gL/gO as well as Antibodies Elicited during Primary HCMV Infection Prevent Epithelial Cell Syncytium Formation [Pathogenesis and Immunity]

  • Human cytomegalovirus (HCMV) may cause disseminated/end-organ disease in congenitally infected newborns and immunosuppressed transplant recipients. Two glycoprotein complexes, gH/gL/gO and gH/gL/pUL128/pUL130/pUL131 (gH/gL/pUL128L; referred to as the pentamer), are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively, in the presence of the viral fusion protein gB. In addition, gH/gL/gO was recently reported to also be required for infection of endothelial/epithelial cells. Virus entry into human fibroblasts involves fusion of the virus envelope with the plasma membrane, whereas entry into endothelial/epithelial cells involves macropinocytosis or endocytosis and low-pH-dependent fusion with endosomes. A large set of neutralizing monoclonal antibodies (MAbs), directed to gH, gB, and multiple components of the pentamer, was developed. In addition, novel anti-gO human monoclonal antibodies were recently isolated. It is known that epithelial cell infection with a wild HCMV strain at a high multiplicity of infection produces a large number of syncytia. Incubation of heavily HCMV VR1814-infected ARPE-19 epithelial cells with neutralizing MAbs to one, two, or three components of the pUL128L portion of the pentamer blocked syncytium formation at an antibody concentration of 10 mmu;g/ml, whereas only a partial inhibitory effect was displayed for MAbs to gO, gH, or gB at the same concentration. A blocking effect was also exhibited by convalescent-phase sera from primary HCMV infections. These findings indicate that the pentamer is required for syncytium formation in epithelial cells.

    IMPORTANCE Human cytomegalovirus (HCMV) mostly infects epithelial and endothelial cells in vivo. Recently, the pentamer protein complex (gH/gL/pUL128L) was identified as being required for infection of these cells, in association with the other protein complex, gH/gL/gO. In primary infections, HCMV migrates to endothelial cells and then to leukocytes, which disseminate the infection throughout the body. The virus then spreads to organs and tissues, mostly infecting either single cells or multinucleated epithelial giant cells (syncytia), depending on the viral load. Potent neutralizing human MAbs directed to distinct binding sites of the pUL128L portion of the pentamer were shown in the past to block virus dissemination. In the present study, MAbs to pUL128L were shown to block syncytium formation with a higher potency than that of MAbs to gO, gH, or gB, thus suggesting their role in limiting virus dissemination. This finding provides additional information useful for the development of anti-HCMV therapeutic antibodies and subunit vaccines.

  • Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins [Virus-Cell Interactions]

  • Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.

    IMPORTANCE Furin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

  • Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets [Pathogenesis and Immunity]

  • Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAaalpha;2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans.

    IMPORTANCE Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

  • Analysis of Conformational Stability of Abnormal Prion Protein Aggregates across the Spectrum of Creutzfeldt-Jakob Disease Prions [Prions]

  • The wide phenotypic variability of prion diseases is thought to depend on the interaction of a host genotype with prion strains that have self-perpetuating biological properties enciphered in distinct conformations of the misfolded prion protein PrPSc. This concept is largely based on indirect approaches studying the effect of proteases or denaturing agents on the physicochemical properties of PrPSc aggregates. Furthermore, most data come from studies on rodent-adapted prion strains, making current understanding of the molecular basis of strains and phenotypic variability in naturally occurring diseases, especially in humans, more limited. To fill this gap, we studied the effects of guanidine hydrochloride (GdnHCl) and heating on PrPSc aggregates extracted from 60 sporadic Creutzfeldt-Jakob disease (CJD) and 6 variant CJD brains. While denaturation curves obtained after exposure of PrPSc to increasing GdnHCl concentrations showed similar profiles among the 7 CJD types analyzed, PrPSc exposure to increasing temperature revealed significantly different and type-specific responses. In particular, MM1 and VV2, the most prevalent and fast-replicating CJD types, showed stable and highly resistant PrPSc aggregates, whereas VV1, a rare and slowly propagating type, revealed unstable aggregates that easily dissolved at low temperature. Taken together, our results indicate that the molecular interactions mediating the aggregation state of PrPSc, possibly enciphering strain diversity, are differently targeted by GdnHCl, temperature, and proteases. Furthermore, the detected positive correlation between the thermostability of PrPSc aggregates and disease transmission efficiency makes inconsistent the proposed hypothesis that a decrease in conformational stability of prions results in an increase in their replication efficiency.

    IMPORTANCE Prion strains are defined as infectious isolates propagating distinctive phenotypic traits after transmission to syngeneic hosts. Although the molecular basis of prion strains is not fully understood, it is largely accepted that variations in prion protein conformation drive the molecular changes leading to the different phenotypes. In this study, we exposed abnormal prion protein aggregates encompassing the spectrum of human prion strains to both guanidine hydrochloride and thermal unfolding. Remarkably, while exposure to increasing temperature revealed significant strain-specific differences in the denaturation profile of the protein, treatment with guanidine hydrochloride did not. The findings suggest that thermal and chemical denaturation perturb the structure of prion protein aggregates differently. Moreover, since the most thermostable prion protein types were those associated with the most prevalent phenotypes and most rapidly and efficiently transmitting strains, the results suggest a direct correlation between strain replication efficiency and the thermostability of prion protein aggregates.

  • Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing [Virus-Cell Interactions]

  • Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4+ T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs.

    IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4+ T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4+ T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4+ T cells will likely dictate different approaches in order to achieve their elimination. For CD4+ T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8+ T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

  • Robust Lys63-Linked Ubiquitination of RIG-I Promotes Cytokine Eruption in Early Influenza B Virus Infection [Virus-Cell Interactions]

  • Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and proinflammatory factors can be induced by influenza B virus rather than influenza A virus infection in alveolar epithelial (A549) cells during early infection. This response is mainly dependent on the retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway. Infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25 and that this interaction blocks the inhibitory effect of the NS1-B C-terminal effector domain (TED) on RIG-I ubiquitination. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins.

    IMPORTANCE Influenza B virus generally causes local mild epidemics but is occasionally lethal to individuals. Existing studies describe the broad characteristics of influenza B virus epidemiology and pathology. However, to develop better prevention and treatments for the disease, determining the concrete molecular mechanisms of pathogenesis becomes pivotal to understand how the host reacts to the challenge of influenza B virus. Thus, we aimed to characterize the host innate immune response to influenza B virus infection. Here, we show that vigorous Lys63-linked ubiquitination of RIG-I and cytokine eruption dependent on RIG-I-mediated signal transduction are induced by virus infection. Additionally, TRIM25 positively regulates RIG-I-mediated signaling by ablating the inhibitory function of NS1-B on RIG-I ubiquitination.

  • Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization [Pathogenesis and Immunity]

  • Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8+ cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8+ cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)hi/phosphorylated AMP-activated protein kinase (AMPK)lo subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control.

    IMPORTANCE In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

  • Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines [Vaccines and Antiviral Agents]

  • Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33ddeg;C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product.

    IMPORTANCE Vaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live-attenuated influenza virus vaccines (LAIVs) based on recombinant viruses whose genomes encode nonoverlapping and independent M1/M2 (split M segment [Ms]) or both M1/M2 and NS1/NEP (Ms and split NS segment [NSs]) open reading frames. Viruses containing a modified M segment were highly attenuated in mice but were able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, the protection efficacy conferred by our viruses with split M segments was better than that conferred by the current temperature-sensitive LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIVs on the basis of the reorganization of spliced viral RNA segments in the genome.

  • Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes [Virus-Cell Interactions]

  • The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH.

    IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.

  • Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles [Vaccines and Antiviral Agents]

  • At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects.

    IMPORTANCE From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV.

  • Nipah Virus C and W Proteins Contribute to Respiratory Disease in Ferrets [Pathogenesis and Immunity]

  • Nipah virus (NiV) is a highly lethal paramyxovirus that recently emerged as a causative agent of febrile encephalitis and severe respiratory disease in humans. The ferret model has emerged as the preferred small-animal model with which to study NiV disease, but much is still unknown about the viral determinants of NiV pathogenesis, including the contribution of the C protein in ferrets. Additionally, studies have yet to examine the synergistic effects of the various P gene products on pathogenesis in animal models. Using recombinant NiVs (rNiVs), we examine the sole contribution of the NiV C protein and the combined contributions of the C and W proteins in the ferret model of NiV pathogenesis. We show that an rNiV void of C expression resulted in 100% mortality, though with limited respiratory disease, like our previously reported rNiV void of W expression; this finding is in stark contrast to the attenuated phenotype observed in previous hamster studies utilizing rNiVs void of C expression. We also observed that an rNiV void of both C and W expression resulted in limited respiratory disease; however, there was severe neurological disease leading to 60% mortality, and the surviving ferrets demonstrated sequelae similar to those for human survivors of NiV encephalitis.

    IMPORTANCE Nipah virus (NiV) is a human pathogen capable of causing lethal respiratory and neurological disease. Many human survivors have long-lasting neurological impairment. Using a ferret model, this study demonstrated the roles of the NiV C and W proteins in pathogenesis, where lack of either the C or the W protein independently decreased the severity of clinical respiratory disease but did not decrease lethality. Abolishing both C and W expression, however, dramatically decreased the severity of respiratory disease and the level of destruction of splenic germinal centers. These ferrets still suffered severe neurological disease: 60% succumbed to disease, and the survivors experienced long-term neurological impairment, such as that seen in human survivors. This new ferret NiV C and W knockout model may allow, for the first time, the examination of interventions to prevent or mitigate the neurological damage and sequelae experienced by human survivors.

  • The Foot-and-Mouth Disease Carrier State Divergence in Cattle [Pathogenesis and Immunity]

  • The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naive or had been vaccinated using a recombinant, adenovirus-vectored vaccine 2 weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% in vaccinated cattle, 67% in nonvaccinated cattle), despite vaccinated cattle having been protected from clinical disease. Analysis of antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days postinfection (dpi) in vaccinated cattle and by 21 dpi in nonvaccinated animals. The anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both the early and the persistent phases of infection. In nonvaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while virus selectively persisted within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and nonvaccinated cattle. FMDV structural and nonstructural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (interferon regulatory factor 7, CXCL10 [gamma interferon-inducible protein 10], gamma interferon, and lambda interferon) in association with persistent FMDV. In contrast, during the transitional phase of infection, the level of expression of IFN- mRNA was higher in follicle-associated epithelium of animals that had cleared the infection. This work provides novel insights into the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis.

    IMPORTANCE The existence of a prolonged, asymptomatic carrier state is a political impediment for control and potential eradication of foot-and-mouth disease (FMD). When FMD outbreaks occur, they are often extinguished by massive depopulation of livestock due to the fear that some animals may have undiagnosed subclinical infection, despite uncertainty over the biological relevance of FMD virus (FMDV) persistence. The work described here elucidates aspects of the FMDV carrier state in cattle which may facilitate identification and/or abrogation of asymptomatic FMDV infection. The divergence between animals that clear infection and those that develop persistent infection was demonstrated to occur earlier than previously established. The host antiviral response in tissues maintaining persistent FMDV was downregulated, whereas upregulation of IFN- mRNA was found in the epithelium of cattle that had recently cleared the infection. This suggests that the clearing of FMDV infection is associated with an enhanced mucosal antiviral response, whereas FMDV persistence is associated with suppression of the host antiviral response.

  • Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly [Structure and Assembly]

  • The structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double-membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability, and maturation. Disturbances of the inter- and intra-TMD interactions of E resulted in the secretion of a larger proportion of capsidless subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in the trans-Golgi network and represent a hallmark of virus maturation.

    IMPORTANCE The assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, noninfectious particles composed of an RNA-containing capsid surrounded by a lipid membrane, with the two integrated envelope proteins, prM and E, arranged in an icosahedral lattice. The mechanism by which the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsidless subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and for the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.

  • Functional Upregulation of the DNA Cytosine Deaminase APOBEC3B by Polyomaviruses [Cellular Response to Infection]

  • The APOBEC3 family of DNA cytosine deaminases has important roles in innate immunity and cancer. It is unclear how DNA tumor viruses regulate these enzymes and how these interactions, in turn, impact the integrity of both the viral and cellular genomes. Polyomavirus (PyVs) are small DNA pathogens that contain oncogenic potentials. In this study, we examined the effects of PyV infection on APOBEC3 expression and activity. We demonstrate that APOBEC3B is specifically upregulated by BK polyomavirus (BKPyV) infection in primary kidney cells and that the upregulated enzyme is active. We further show that the BKPyV large T antigen, as well as large T antigens from related polyomaviruses, is alone capable of upregulating APOBEC3B expression and activity. Furthermore, we assessed the impact of A3B on productive BKPyV infection and viral genome evolution. Although the specific knockdown of APOBEC3B has little short-term effect on productive BKPyV infection, our informatics analyses indicate that the preferred target sequences of APOBEC3B are depleted in BKPyV genomes and that this motif underrepresentation is enriched on the nontranscribed stand of the viral genome, which is also the lagging strand during viral DNA replication. Our results suggest that PyV infection upregulates APOBEC3B activity to influence virus sequence composition over longer evolutionary periods. These findings also imply that the increased activity of APOBEC3B may contribute to PyV-mediated tumorigenesis.

    IMPORTANCE Polyomaviruses (PyVs) are a group of emerging pathogens that can cause severe diseases, including cancers in immunosuppressed individuals. Here we describe the finding that PyV infection specifically induces the innate immune DNA cytosine deaminase APOBEC3B. The induced APOBEC3B enzyme is fully functional and therefore may exert mutational effects on both viral and host cell DNA. We provide bioinformatic evidence that, consistent with this idea, BK polyomavirus genomes are depleted of APOBEC3B-preferred target motifs and enriched for the corresponding predicted reaction products. These data imply that the interplay between PyV infection and APOBEC proteins may have significant impact on both viral evolution and virus-induced tumorigenesis.

  • Hepatitis C Virus-Induced Upregulation of MicroRNA miR-146a-5p in Hepatocytes Promotes Viral Infection and Deregulates Metabolic Pathways Associated with Liver Disease Pathogenesis [Virus-Cell Interactions]

  • Hepatitis C virus (HCV)-induced chronic liver disease is a leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in homeostasis within the liver, and deregulation of miRNAs has been associated with liver disease, including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here we combined a hepatocyte-like cell-based model system, high-throughput small RNA sequencing, computational analysis, and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least 2-fold, including miRNAs that were previously described to target genes associated with inflammation, fibrosis, and cancer development. Further investigation demonstrated that the miR-146a-5p level was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes, as well as in liver tissue from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p overexpression modulates pathways that are related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p has a positive impact on late steps of the viral replication cycle, thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease.

    IMPORTANCE HCV is a leading cause of chronic liver disease and cancer. However, how HCV induces liver cancer remains poorly understood. There is accumulating evidence that a viral cure does not eliminate the risk for HCC development. Thus, there is an unmet medical need to develop novel approaches to predict and prevent virus-induced HCC. miRNA expression is known to be deregulated in liver disease and cancer. Furthermore, miRNAs are essential for HCV replication, and HCV infection alters miRNA expression. However, how miRNAs contribute to HCV-driven pathogenesis remains elusive. Here we show that HCV induces miRNAs that may contribute to liver injury and carcinogenesis. The miR-146a-5p level was consistently increased in different cell-based models of HCV infection and in HCV patient-derived liver tissue. Furthermore, miR-146a-5p increased HCV infection. Collectively, our data are relevant to understanding viral pathogenesis and may open perspectives for novel biomarkers and prevention of virus-induced liver disease and HCC.

  • Unexpected Interfarm Transmission Dynamics during a Highly Pathogenic Avian Influenza Epidemic [Genetic Diversity and Evolution]

  • Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain.

    IMPORTANCE The within- and between-host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within- and between-farm genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic form to the highly pathogenic form occurred within the first infected flock, where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

  • Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis [Virus-Cell Interactions]

  • The pathogenic Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with a high rate of mortality in humans. Several LASV receptors, including dystroglycan (DG), TAM receptor tyrosine kinases, and C-type lectins, have been identified, suggesting complex receptor use. Upon receptor binding, LASV enters the host cell via an unknown clathrin- and dynamin-independent pathway that delivers the virus to late endosomes, where fusion occurs. Here we investigated the mechanisms underlying LASV endocytosis in human cells in the context of productive arenavirus infection, using recombinant lymphocytic choriomeningitis virus (rLCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that rLCMV-LASVGP entered human epithelial cells via DG using a macropinocytosis-related pathway independently of alternative receptors. Dystroglycan-mediated entry of rLCMV-LASVGP required sodium hydrogen exchangers, actin, and the GTPase Cdc42 and its downstream targets, p21-activating kinase-1 (PAK1) and Wiskott-Aldrich syndrome protein (N-Wasp). Unlike other viruses that enter cells via macropinocytosis, rLCMV-LASVGP entry did not induce overt changes in cellular morphology and hardly affected actin dynamics or fluid uptake. Screening of kinase inhibitors identified protein kinase C, phosphoinositide 3-kinase, and the receptor tyrosine kinase human hepatocyte growth factor receptor (HGFR) to be regulators of rLCMV-LASVGP entry. The HGFR inhibitor EMD 1214063, a candidate anticancer drug, showed antiviral activity against rLCMV-LASVGP at the level of entry. When combined with ribavirin, which is currently used to treat human arenavirus infection, EMD 1214063 showed additive antiviral effects. In sum, our study reveals that DG can link LASV to an unusual pathway of macropinocytosis that causes only minimal perturbation of the host cell and identifies cellular kinases to be possible novel targets for therapeutic intervention.

    IMPORTANCE Lassa virus (LASV) causes several hundred thousand infections per year in Western Africa, with the mortality rate among hospitalized patients being high. The current lack of a vaccine and the limited therapeutic options at hand make the development of new drugs against LASV a high priority. In the present study, we uncover that LASV entry into human cells via its major receptor, dystroglycan, involves an unusual pathway of macropinocytosis and define a set of cellular factors implicated in the regulation of LASV entry. A screen of kinase inhibitors revealed HGFR to be a possible candidate target for antiviral drugs against LASV. An HGFR candidate inhibitor currently being evaluated for cancer treatment showed potent antiviral activity and additive drug effects with ribavirin, which is used in the clinic to treat human LASV infection. In sum, our study reveals novel fundamental aspects of the LASV-host cell interaction and highlights a possible candidate drug target for therapeutic intervention.

  • Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV), a betaherpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins that mediate binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown. In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells by use of a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five proviral host factors from different protein families and eight antiviral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel proviral host factor and was analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either broad or narrow cell tropism were equally impaired. Infection of CD151-depleted cells by a fluorescent virus with differentially labeled capsid and envelope proteins revealed a role of CD151 in viral penetration but not in adsorption to the cell. In conclusion, the tetraspanin CD151 has emerged as a novel host factor in HCMV entry and as a putative antiviral target.

    IMPORTANCE At present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with putative roles in this process were identified. The tetraspanin CD151 was discovered as a previously unrecognized proviral host factor for HCMV and was found to support viral penetration into the target cells. The findings of this study shed light on the cellular contribution during the initial steps of HCMV infection and open a new direction in HCMV research.

  • Human Cytomegalovirus Induces an Atypical Activation of Akt To Stimulate the Survival of Short-Lived Monocytes [Cellular Response to Infection]

  • Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110 isoform, which was required for the viability of uninfected monocytes, to the p110bbeta; isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy.

    IMPORTANCE HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.

  • Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses [Cellular Response to Infection]

  • Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development.

    IMPORTANCE RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.

  • CD69 Deficiency Enhances the Host Response to Vaccinia Virus Infection through Altered NK Cell Homeostasis [Pathogenesis and Immunity]

  • During the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69nndash;/nndash; mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69nndash;/nndash; lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival.

    IMPORTANCE We show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.

  • NF-{kappa}B Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-B (NF-B) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-B binding sites abolished activation of BART promoters by NF-B. Binding of p50 subunits to NF-B sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-B activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IB kinase inhibitor PS-1145 inhibited NF-B activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-B activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-B signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-B activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC.

    IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-B signaling. EBV LMP1 is a potent activator of NF-B signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-B signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-B signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-B signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

  • Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage [Cellular Response to Infection]

  • In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2aalpha;, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway.

    IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6Pro. This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

  • In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation [Structure and Assembly]

  • Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membrane-spanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly.

    IMPORTANCE Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.

  • Suppression of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by 5'-AMP-Activated Protein Kinase [Virus-Cell Interactions]

  • The host intracellular antiviral restriction factors inhibit viral infection and replication. The 5'-AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. Activated AMPK inhibits the replication of numerous RNA viruses but enhances the entry of vaccinia virus. However, the role of AMPK in herpesvirus infection is unclear. In this study, we showed that the constitutive AMPK activity restricted Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in primary human umbilical vein endothelial cells while KSHV infection did not markedly affect the endogenous AMPK activity. Knockdown of the AMPKaalpha;1 considerably enhanced the expression of viral lytic genes and the production of infectious virions, while overexpression of a constitutively active AMPK had the opposite effects. Accordingly, an AMPK inhibitor, compound C, augmented viral lytic gene expressions and virion productions but an AMPK agonist, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), suppressed both. Furthermore, a common diabetes drug, metformin, which carries an AMPK-agonistic activity, drastically inhibited the expression of viral lytic genes and the production of infectious virions, suggesting the use of metformin as a therapeutic agent for KSHV infection and replication. Together, these results identify the host AMPK as a KSHV restriction factor that can serve as a potential therapeutic target.

    IMPORTANCE Host cells encode specific proteins to restrict viral infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. In this study, we have identified 5'-AMP-activated protein kinase (AMPK), a cellular energy sensor, as a restriction factor of KSHV lytic replication during primary infection. Activation of AMPK suppresses, while inhibition of AMPK enhances, KSHV lytic replication by regulating the expression of viral genes. AICAR and metformin, both of which are AMPK agonists currently used in clinics for the treatment of conditions associated with metabolic disorders, inhibit KSHV lytic replication. Thus, our work has identified AMPK as a potential therapeutic target and AICAR and metformin as potential therapeutic agents for KSHV-associated cancers.

  • Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication [Genome Replication and Regulation of Viral Gene Expression]

  • An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae.

    IMPORTANCE Recently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a better general understanding of virus-encoded miRNAs. In brief, our study implied that AcMNPV-miR-1 constrains viral replication and cellular infection but enhances larval infection.

  • The Emerging Duck Flavivirus Is Not Pathogenic for Primates and Is Highly Sensitive to Mammalian Interferon Antiviral Signaling [Pathogenesis and Immunity]

  • Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus.

    IMPORTANCE Mosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile, and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus, DTMUV (also known as duck egg drop syndrome flavivirus [DEDSV]) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other nonavian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.

  • Association between Hemagglutinin Stem-Reactive Antibodies and Influenza A/H1N1 Virus Infection during the 2009 Pandemic [Vaccines and Antiviral Agents]

  • The discovery of influenza virus broadly neutralizing (BrN) antibodies prompted efforts to develop universal vaccines. Influenza virus stem-reactive (SR) broadly neutralizing antibodies have been detected by screening antibody phage display libraries. However, studies of SR BrN antibodies in human serum, and their association with natural infection, are limited. To address this, pre- and postpandemic sera from a prospective community cohort study in Vietnam were assessed for antibodies that inhibit SR BrN monoclonal antibody (MAb) (C179) binding to H1N1 pandemic 2009 virus (H1N1pdm09). Of 270 households, 33 with at least one confirmed H1N1pdm09 illness or at least two seroconverters were included. The included households comprised 71 infected and 41 noninfected participants. Sera were tested as 2-fold dilutions between 1:5 and 1:40. Fifty percent C179 inhibition (IC50) titers did not exceed 10, although both IC50 titers and percent C179 inhibition by sera diluted 1:5 or 1:10 correlated with hemagglutination inhibition (HI) and microneutralization (MN) titers (all P llt; 0.001). Thirteen (12%) participants had detectable prepandemic IC50 titers, but only one reached a titer of 10. This proportion increased to 44% after the pandemic, when 39 participants had a titer of 10, and 67% of infected compared to 44% of noninfected had detectable IC50 titers (P llt; 0.001). The low levels of SR antibodies in prepandemic sera were not associated with subsequent H1N1pdm09 infection (P = 0.241), and the higher levels induced by H1N1pdm09 infection returned to prepandemic levels within 2 years. The findings indicate that natural infection induces only low titers of SR antibodies that are not sustained.

    IMPORTANCE Universal influenza vaccines could have substantial health and economic benefits. The focus of universal vaccine research has been to induce antibodies that prevent infection by diverse influenza virus strains. These so-called broadly neutralizing antibodies are readily detected in mice and ferrets after infection with a series of distinct influenza virus strains. The 2009 H1N1 pandemic provided an opportunity to investigate whether infection with a novel strain induced broadly neutralizing antibodies in humans. We found that broadly neutralizing antibodies were induced, but levels were low and poorly maintained. This could represent an obstacle for universal vaccine development and warrants further investigation.

  • Both CD8+ and CD4+ T Cells Contribute to Corneal Clouding and Viral Clearance following Vaccinia Virus Infection in C57BL/6 Mice [Pathogenesis and Immunity]

  • Vaccinia virus (VACV) keratitis is a serious complication following smallpox vaccination and can lead to blindness. The pathological mechanisms involved in ocular VACV infection are poorly understood. Previous studies have used rabbits, but the lack of immune reagents and transgenic or knockout animals makes them less suitable for mechanistic studies. We report that infection of C57BL/6 mice with 1 x 107 PFU of vaccinia virus strain WR results in blepharitis, corneal neovascularization, and stromal keratitis. The DryVax strain of VACV was completely attenuated. Infection required corneal scarification and replication-competent virus, and the severity of ocular disease was similar in 4- to 6-week-old and 1-year-old mice. Viral titers peaked at approximately 1 x 106 PFU on day 5 postinfection, and virus had not cleared by day 13 postinfection. Neutrophils were found in the peripheral cornea on day 1 after infection and then declined, followed by infiltration of both CD4+ and CD8+ T cells, which remained peripheral throughout the infection. Blood vessel growth extended 2 to 5 mm into the cornea from the limbus. Infection of CD4nndash;/nndash;, CD8nndash;/nndash;, or antibody-depleted mice resulted in similar disease severity and corneal clouding, indicating that both T-cell subsets were involved in the immunopathological response. Depletion of both CD4+ and CD8+ T cells resulted in significantly more severe disease and failure to clear the virus. On the basis of our results, the pathology of VACV keratitis is significantly different from that of herpes simplex virus keratitis. Further studies are likely to reveal novel information regarding virulence and immune responses to viral ocular infection.

    IMPORTANCE Potentially blinding eye infections can occur after vaccination for smallpox. Very little is known about the pathological mechanisms that are involved, and the information that is available was generated using rabbit models. The lack of immunological reagents for rabbits makes such studies difficult. We characterized a mouse model of vaccinia virus ocular disease using C57BL/6 mice and strain WR and show that both CD4+ and CD8+ T-cell subsets play a role in the blinding eye disease and in controlling virus replication. On the basis of these results, vaccinia virus keratitis is significantly different from herpes simplex virus keratitis, and further studies using this model should generate novel insights into immunopathological responses to viral ocular infection.

  • Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response [Virus-Cell Interactions]

  • Bats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro. These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-X), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-X). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-X replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-B. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential for in vitro viral replication.

    IMPORTANCE Bats harbor genetically diverse SARS-like coronaviruses (SL-CoVs), and some of them have the potential for interspecies transmission. A unique open reading frame (ORFX) was identified in the genomes of two recently isolated bat SL-CoV strains (WIV1 and -16). It will therefore be critical to clarify whether and how this protein contributes to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene that is involved in the modulation of the host immune response but is not essential for in vitro viral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for study of the pathogenesis of this group of viruses and to develop therapeutics for future control of emerging SARS-like infections.

  • HIV-1 Gag, Envelope, and Extracellular Determinants Cooperate To Regulate the Stability and Turnover of Virological Synapses [Virus-Cell Interactions]

  • Retroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high-avidity Env-CD4 linkages are resolved over time is unknown. We describe here a tractable two-color, long-term (ggt;24 h) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-to-cell transmission wherein (i) Env-receptor interactions anchor target cells to infected cells, (ii) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and (iii) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes.

    IMPORTANCE HIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses (VSs). The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals. We describe here a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people.

  • MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus [Virus-Cell Interactions]

  • Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction.

    IMPORTANCE We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

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  • Nuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated human Herpesvirus 8 (KSHV) and readily infects cells of both human and rhesus monkey origin. We used the CRISPR:Cas9 technique to generate knockout (ko) cells for each of the four ND10 components PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blot. Knock-out of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and loss of ND10 structures, whereas protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition of de-novo gene expression prevented loss of SP100 and PML in cells that do not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein.

    IMPORTANCE Our findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gamma-herpesviruses as important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then PML with a delayed kinetic, which clearly differs from other gamma-herpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the former two are targeted for degradation by RRV, and the latter still potently restricts replication of RRV.

  • The MHC II HLA-DR{alpha} is Downregulated by KSHV encoded lytic transactivator RTA and MARCH8 [PublishAheadOfPrint]

  • Kaposi's-sarcoma associated herpesvirus (KSHV) maintains two modes of life cycle, latency and lytic phase. To evade the attack of cell host immune system, KSHV switches from lytic to latency, a phase where only a few of viral proteins are expressed. The mechanism by which KSHV evades the attack of the immune system and establishes latency has not been fully understood. MHC-II molecules are key componments of the immune system defense mechanism against viral infections. Here we report that the HLA-DRaalpha;, a member of the MHC II molecules, was downregulated by the replication and trancription activator (RTA) protein encoded by KSHV ORF50, an important regulator of the viral life cycle. RTA not only downregulated HLA-DRaalpha; at the protein level through direct binding and degradation through the proteasome pathway, but also indirectly down regulated the protein level of HLA-DR aalpha; by enchancing the expression of MARCH8, a member of the membrane-associated RING-CH (MARCH) proteins. Our findings indicate that KSHV RTA facilitates evasion of the virus from the immune system through manipulation of HLA-DRaalpha;.

    Importance Kaposi's sarcoma-associated herpesvirus has a causal role in a number of human cancers and its persistence in infected cells is controlled by the host's immune system. The mechanism by which KSHV evades an attack by immune system has not been well understood. This work represents studies which identifies a novel mechanism by which the virus can facilitate evasion of an immune system. We now show that RTA, the replication and transcription activator encoded by KSHV (ORF50) can function as an E3 ligase to degrade HLA-DRaalpha;. It can directly bind and induce degradation of the HLA-DRaalpha; through the ubiquitin-proteasomal degradation pathway. In addition to the direct regulation of HLA-DRaalpha;, RTA can also indirectly down-regulate the level of HLA-DRaalpha; protein by upregulating transcription of MARCH8. Increased MARCH8 results in the downregulation of HLA-DRaalpha;. Furthermore we also demonstrated that expression of HLA-DRaalpha; was impaired in KSHV de novo infection.

  • Flow Cytometric and Cytokine ELISpot Approaches to Characterize the Cell-mediated Immune Response in Ferrets Following Influenza Virus Infection [PublishAheadOfPrint]

  • Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this report, we developed flow cytometric and T cell ELISpot approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multi-parameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells and MHC class II+ APCs both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered 10 days post-infection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen-specificity of influenza-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1 and NP proteins and that total reactivity to influenza post-infection represented approximately 0.1% of the circulating PBMCs. Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.

    IMPORTANCE Ferrets are an ideal animal model to study transmission, disease and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which was overlayed with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest there is considerable heterogeneity at the MHC locus, akin to humans and an area of intense research interest.

  • Kynurenine Reduces Memory CD4 T-cell Survival by Interfering with IL-2 Signaling early During HIV-1 Infection [PublishAheadOfPrint]

  • Early HIV-1 infection is characterized by enhanced tryptophan catabolism, which contributes to immune suppression and disease progression. However, mechanism by which kynurenine, a tryptophan-related metabolite, induces immune suppression remains poorly understood. Herein, we showed that the increased production of kynurenine correlates with defective IL-2 signaling in memory CD4 T-cells from HIV-infected subjects. Defective IL-2 signaling in these subjects, which drives reduced protection from Fas-mediated apoptosis, was also associated with memory CD4 T-cell loss. Treatment of memory CD4 T-cells with plasma concentration of kynurenine inhibits IL-2 signaling through the production of reactive oxygen species. We further showed that IL-2 signaling in memory CD4 T-cells was improved by the antioxidant N-acetylcysteine. Early initiation of antiretroviral therapy restored IL-2 response in memory CD4 T-cells by reducing ROS and kynurenine production. Study findings provide a kynurenine-dependant mechanism though IL-2 signaling for the reduced CD4 T cell survival that can be reversed by early treatment initiation in HIV-1 infection.

    IMPORTANCE The persistence of functional memory CD4 T-cells represents the basis for a long-lasting immune protection in individuals after exposure to HIV-1. Unfortunately, primary HIV-1 infection results in the massive loss of these cells within weeks of infection which is mainly driven by inflammation and massive infection by the virus. These new findings show that the enhanced production of kynurenine, a metabolite related to the tryptophan catabolism, also impairs memory CD4 T-cell survival and interferes with IL-2 signaling early during HIV-1 infection.

  • Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses that Protect Mice from Lethal Challenge [PublishAheadOfPrint]

  • Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide and causes respiratory disease, abortion, and in some cases, neurological disease. EHV-1 strain KyA is attenuated in the mouse and equine, whereas wild-type strain RacL11 induces severe inflammation of the lung, causing infected mice to succumb at 4 to 6 days post-infection. Our previous results showed that KyA immunization protected CBA mice from pathogenic RacL11 challenge at 2 and 4 weeks post-immunization, and that the KyA infection elicited protective humoral and cell-mediated immune responses. To investigate the protective mechanisms of KyA by innate immune responses, KyA-immunized mice were challenged with RacL11 at various times post-vaccination. KyA immunization protected mice from RacL11 challenge at 1 to 7 days post-immunization. Immunized mice lost less than 10% of their body weight and rapidly regained weight. Lung virus titers in KyA-immunized mice were 1,000-fold lower at 2 days post-RacL11 challenge than titers in lungs of non-immunized mice, indicative of accelerated virus clearance. Affymetrix microarray analysis revealed that IFN- and 16 antiviral interferon-stimulated genes (ISGs) were upregulated 3.1 to 48.2-fold at 8 h post-challenge in the lungs of RacL11-challenged mice that had been immunized with KyA. Murine IFN- inhibited EHV-1 infection of murine alveolar macrophage cells and protected mice against lethal EHV-1 challenge, suggesting that IFN- expression is important in mediating protection elicited by KyA immunization. These results suggest that EHV-1 KyA may be used as a live attenuated EHV-1 vaccine as well as a prophylactic agent in horses.

    IMPORTANCE Viral infection of cells initiates a signal cascade of events that ultimately attempts to limit viral replication and prevent infection through the expression of host antiviral proteins. Here, we show that EHV-1 KyA immunization effectively protected CBA mice from pathogenic RacL11 challenge at 1 to 7 days post-vaccination, and increased expression of IFN- and 16 antiviral interferon-stimulated genes (ISG). The administration of IFN- blocked EHV-1 replication in murine alveolar macrophages and mouse lungs and protected mice from lethal challenge. To our knowledge, this is the first report of an attenuated EHV-1 vaccine that protects the animal at 1 to 7 days post-immunization by innate immune responses. Our findings suggested that IFN- serves as a novel prophylactic agent and may offer new strategies for the development of anti-EHV-1 agents in the equine.

  • In vivo examination of mouse APOBEC3- and human APOBEC3A- and APOBEC3G-mediated restriction of parvovirus and herpesvirus infection in mouse models [PublishAheadOfPrint]

  • APOBEC3 knockout and human APOBEC3A and 3G transgenic mice were tested for their ability to be infected by the herpesviruses Herpes Simplex Virus 1 and Murine Herpes Virus 68, and the parvovirus Minute Virus of Mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic and wild type mice were equally infected by the herpesviruses while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo.

    IMPORTANCE It is well-established that APOBEC3 proteins in different species are a critical component of the host anti-retroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly anti-viral and demonstrate the importance of testing their activity in vivo.

  • The Evolutionary Histories of Antiretroviral Proteins SERINC3 and SERINC5 Do Not Support an Evolutionary Arms Race in Primates [PublishAheadOfPrint]

  • Molecular evolutionary arms races between viruses and their hosts are important drivers of adaptation. These Red Queen dynamics have been frequently observed in primate retroviruses and their antagonists, host restriction factor genes, such as APOBEC3F/G, TRIM5-aalpha;, SAMHD1, and BST-2. Host restriction factors have experienced some of the most intense and pervasive adaptive evolution documented in primates. Recently, two novel host factors, SERINC3 and SERINC5, were identified as the targets of HIV-1 Nef, a protein crucial for the optimal infectivity of virus particles. Here, we compared the evolutionary fingerprints of SERINC3 and SERINC5 to those of other primate restriction factors and to a set of other genes with diverse functions. SERINC genes evolved in a manner distinct from the canonical arms race dynamics seen in the other restriction factors. Despite their antiviral activity against HIV-1 and other retroviruses, SERINC3 and SERINC5 have a relatively uneventful evolutionary history in primates.

    Importance Restriction factors are host proteins that block viral infection and replication. Many viruses, like HIV-1 and related retroviruses, evolved accessory proteins to counteract these restriction factors. The importance of these interactions is evidenced by the intense adaptive selection pressures that dominates the evolutionary histories of both the host and viral genes involved in this so-called arms race. The dynamics of these arms races can point to mechanisms by which these viral infections can be prevented. Two human genes were recently identified as targets of an HIV-1 accessory protein important for viral infectivity: SERINC3 and SERINC5. Unexpectedly, we found that these SERINC genes, unlike other host restriction factors, show no evidence of a recent evolutionary arms race with viral pathogens.

  • The influenza A virus genotype determines the anti-viral function of NF-{kappa}B [PublishAheadOfPrint]

  • The role of NF-B for influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also anti-viral functions of this transcription factor have been described. To address this issue, we used CRISPR-Cas9-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-B pathway. Cells devoid of either the central NF-B essential modulator (NEMO) scaffold protein and thus defect in IB kinase (IKK) activation or cells not expressing the NF-B DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range and its mouse-adapted variant SC35M. While NF-B was not relevant for replication of SC35M, the absence of NF-B activity increased replication of the non-adapted SC35. This anti-viral effect of NF-B was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. Defect NF-B signaling resulted in diminished IAV-triggered phosphorylation of IRF3 and expression of the anti-viral IFNbbeta; gene. To identify the viral proteins responsible for NF-B-dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-B, emphasizing the importance of the viral genotype for susceptibility to the anti-viral functions of NF-B.

    IMPORTANCE This study addresses two different issues: First, we investigated the role of the host cell transcription factor NF-B for IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-B displays an anti-viral function, and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-B function greatly improved the growth of a non-adapted IAV, suggesting that NF-B may contribute to the maintenance of the host species barrier.

  • Structure-Function Analysis of the {Phi}X174 DNA Piloting Protein using Length Altering Mutations [PublishAheadOfPrint]

  • Although the X174 H protein is monomeric during procapsid morphogenesis, ten proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation is unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for their ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they were either not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85 amino acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein.

    IMPORTANCE The H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the aalpha;-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long aalpha;-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using inframe insertions and deletions as presented herein.

  • Distinct particle morphologies revealed through comparative parallel analyses of retrovirus-like particles [PublishAheadOfPrint]

  • The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate nndash; in parallel comparative analyses nndash; Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP) created from all representative retroviral genera: alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumaretrovirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative beta- and epsilonretrovirus Gag proteins. This is the first report of epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative beta- and epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and spumaretrovirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway.

    Importance: Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis nndash; including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1). Here in this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway.

  • Establishment and Reversal of HIV-1 Latency in Naïve and Central Memory CD4+ T Cells in Vitro [PublishAheadOfPrint]

  • The latent HIV-1 reservoir primarily resides in resting CD4+ T cells which is a heterogeneous population composed of both naïve (TN) and memory cells. In HIV-1-infected individuals, viral DNA has been detected in both naïve and memory CD4+ T cell subsets, although the frequency of HIV-1 DNA is typically higher in memory cells, particularly in the central memory (TCM) cell subset. TN and TCM cells are distinct cell populations distinguished by many phenotypic and physiological differences. In this study we used a primary cell model of HIV-1 latency that utilizes direct infection of highly purified TN and TCM cells to address differences in the establishment and reversal of HIV-1 latency. Consistent with what is seen in vivo, we found that HIV-1 infected TN cells less efficiently than TCM cells. However, when the infected TN cells were treated with latency reversing agents, including anti-CD3/CD28 antibodies, phorbol-myristate-acetate/phytohaemagglutinin and prostratin, as much, if not more, extracellular virion-associated HIV-1 RNA was produced per infected TN cell than infected TCM cell. There were no major differences in the genomic distribution of HIV-1 integration sites between TN and TCM cells that accounted for these observed differences. We observed decay of the latent HIV-1 cells in both T cell subsets after exposure to each of the latency reversing agents. Collectively, these data highlight significant differences in the establishment and reversal of HIV-1 latency in TN and TCM CD4+ T cells, and suggest that each subset should be independently studied in pre-clinical and clinical studies.

    IMPORTANCE The latent HIV-1 reservoir is frequently described as residing within resting memory CD4+ T cells. This is largely due to the consistent finding that memory CD4+ T cells, specifically the central (TCM) and transitional memory compartments, harbor the highest levels of HIV-1 DNA in individuals on suppressive therapy. This has yielded little research into the contribution of CD4+ naïve (TN) cells to the latent reservoir. In this study, we show that although TN cells harbor significantly lower levels of HIV-1 DNA, following latency reversal, they produced as much, if not more, virions than did the TCM cells. This suggests that latently infected TN cells may be a major source of virus following treatment interruption or failure. These findings highlight the need for a better understanding of the establishment and reversal of HIV-1 latency in TN cells when evaluating therapeutic approaches to eliminate the latent reservoir.

  • Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA Packaging Motors with Revolving Mechanism [PublishAheadOfPrint]

  • The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate development of new potent antiviral drugs. Recently, a method has been described for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential action mechanism. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA packaging motors. Inhibiting multi-subunit targets with sequential action resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral dsDNA packaging motor with sequential one-way revolving will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors were classified into two categories: linear and rotation motion. Recently discovered was a third type of biomotor, including the viral DNA packaging motor, that use a revolving mechanism without rotation. By analogy, rotation resembles the Earth rotating on its own axis, while revolving resembles the Earth revolving around the Sun (see animations: Herein, we review the structures of viral dsDNA packaging motors, the stoichiometry of the motor components, and the motion mechanism of the motors. All viral dsDNA packaging motors contain a high stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometry of target complexes that work sequentially.

  • Human Th17 cells lack HIV-inhibitory RNases and are highly permissive to productive HIV infection [PublishAheadOfPrint]

  • Human Immunodeficiency Virus (HIV) infects and depletes CD4+ T cells, but subsets of CD4+ T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes IL-17-producing "T helper 17" (Th17) cells and "T follicular helper" (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation - a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4+ T cells were sorted, activated in Th17- or Th0-polarizing conditions and infected, then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells compared with IL-17- or IFN+ cells, even upon infecting with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of Ribonuclease A superfamily proteins, and report for the first time that RNase6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication.

    IMPORTANCE Our study compares the intracellular replicative capacity of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity, and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4+ T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells.

    Characterizing cells that support high levels of viral replication (rather than becoming latently infected or undergoing cell death) informs the search for new therapeutics aimed at manipulating intracellular signaling pathways and/or transcriptional factors that affect HIV replication.

  • cGAS-STING Signaling Regulates Initial Innate Control of Cytomegalovirus Infection [PublishAheadOfPrint]

  • Several innate sensing pathways contribute to control of early cytomegalovirus (CMV) infection, leading to a multi-phasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells, however the initial burst of IFN-I that occurs within the first few hours in vivo is TLR-independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING and IRF3 signaling. Disruption of STING expression in endothelial cells by CRISPR-Cas9 revealed it is essential for the induction of IFN-I and restricting CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in non-hematopoietic cells, and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo.

    Importance: Cytomegalovirus (CMV) is one of the most common viral pathogens, with a majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immune compromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPR revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice STING is necessary for the first-phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection.

  • Adeno-Associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Co-Infected with HSV-1 Following Peripheral Inoculation [PublishAheadOfPrint]

  • Following infection of epithelial tissues, Herpes Simplex Virus type 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with varying amounts of rAAV as well as rAAV capsid mutants. We demonstrate that this method of inoculation can achieve transduction of ggt;90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we show that corneal inoculation with rAAV vectors in the rabbit efficiently transduces ggt;70% of the TG neurons in the optic tract. Finally, we demonstrate that co-infection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 results in co-localization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV or sensory ganglia dysfunctions.

    IMPORTANCE Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglia sensory neurons following direct intraganglionic sciatic nerve injection, intraperitoneal and intravenous injection, as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally, at an epithelial surface that has been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to co-transduce HSV-1 infected neurons both in the mouse and rabbit opens the opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency and to reactivation from latency in vivo.

  • BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating TNF{alpha} [PublishAheadOfPrint]

  • Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increased secretion of several inflammatory factors, whereas lytic infections evade the antiviral inflammatory response. However, the changes and roles of the inflammatory microenvironment during the switch between EBV lifecycles remain unknown. In the present study, we demonstrate that latent EBV infection in EBV-positive cells triggers SASP in neighboring epithelial cells. By contrast, lytic EBV infection abolishes this phenotype, BZLF1 attenuates the transmission of paracrine senescence during lytic EBV infection by down-regulating TNFaalpha; secretion. A mutant BZLF1 protein, BZLF1207-210, that cannot inhibit TNFaalpha; secretion while maintaining viral transcription, fails to block paracrine senescence, whereas a neutralizing antibody against TNFaalpha; is sufficient to restore its inhibition. Furthermore, latent EBV infection induces oxidative stress in neighboring cells, while BZLF1-mediated downregulation of TNFaalpha; reduces reactive oxygen species (ROS) levels in neighboring cells, and ROS scavengers alleviate paracrine senescence. These results suggest that lytic EBV infection attenuates the transmission of inflammatory paracrine senescence through BZLF1-downregulating TNFaalpha; secretion and alters the inflammatory microenvironment to allow virus propagation and persistence.

    IMPORTANCE The senescence-associated secretory phenotype (SASP), an important tumorigenic process, is triggered and transmitted by inflammatory factors. The different life cycles of Epstein-Barr virus (EBV) infection in EBV-positive cells employ distinct strategies to modulate the inflammatory response and senescence. The elevation of inflammatory factors during latent EBV infection promotes SASP in uninfected cells. By contrast, during the viral lytic cycle, BZLF1 suppresses the production of TNFaalpha;, resulting in the attenuation of paracrine inflammatory senescence. This finding indicates that EBV evades inflammatory senescence during lytic infection and switches from facilitating tumor-promoting SASP to generating a virus-propagating microenvironment, thereby facilitating viral spread in EBV-associated diseases.

  • HSV-2 Virion Host Shutoff Endoribonuclease Activity is Required to Disrupt Stress Granule Formation [PublishAheadOfPrint]

  • We previously established that cells infected with HSV-2 are disrupted in their ability to form stress granules (SGs) in response to oxidative stress and that this disruption is mediated by virion host shutoff protein (vhs), a virion associated endoribonuclease. Here, we test the requirement for vhs endoribonuclease activity in disruption of SG formation. We analyzed the ability of HSV-2 vhs carrying the point mutation D215N, which ablates its endoribonuclease activity, to disrupt SG formation in both transfected and infected cells. We present evidence that ablation of vhs endoribonuclease activity results in defects in vhs-mediated disruption of SG formation. Furthermore, we demonstrate that preformed SGs can be disassembled by HSV-2 infection in a manner that requires vhs endoribonuclease activity and that, befitting this ability to promote SG disassembly, vhs is able to localize to SGs. Taken together, these data indicate that endoribonuclease activity must be maintained in order for vhs to disrupt SG formation. We propose a model whereby vhs-mediated destruction of SG mRNA promotes SG disassembly and may also prevent SG assembly.

    IMPORTANCE Stress granules (SGs) are transient cytoplasmic structures that form when a cell is exposed to stress. SGs are emerging as potential barriers to viral infection, necessitating a more thorough understanding of their basic biology. We identified virion host shutoff protein (vhs) as a herpes simplex virus 2 (HSV-2) protein capable of disrupting SG formation. As mRNA is a central component of SGs and the best-characterized activity of vhs is as an endoribonuclease specific for mRNA in vivo, we investigated the requirement for vhs endoribonuclease activity in disruption of SG formation. Our studies demonstrate that endoribonuclease activity is required for vhs to disrupt SG formation and, more specifically, that SG disassembly can be driven by vhs endoribonuclease activity. Notably, during the course of these studies we discovered that there is an ordered departure of SG components during their disassembly and furthermore, that vhs itself has the capacity to localize to SGs.

  • Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane Proximal Stalk Region Influences Stability, Receptor Binding and Neuraminidase Activity [PublishAheadOfPrint]

  • Paramyxoviridae consist of a large family of enveloped, negative sense, non-segmented single stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN; also called H or G depending on virus type and substrate) which acts as a receptor binding protein and a fusion (F) protein which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN four helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross linking as well as functional assays to investigate the role of the structurally unresolved membrane proximal stalk region (MPSR) (residues 37-58) of HN in the context of headless and full length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F-triggering.

    IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F) to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane anchored flexible stalk of ~80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane proximal stalk region (MPSR) (HN, residues 37-56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.

  • Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR/Cas9 Knock-Out Cell Lines and Vaccinia Virus Mutants [PublishAheadOfPrint]

  • Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent dsRNA accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 co-localized within cytoplasmic viral factories in cells infected with a decappng enzyme mutant as well as wild-type VACV and they co-precipitated with antibody. An E3 deletion mutant induced PKR and eIF2aalpha; phosphorylation earlier and more strongly than a decapping enzyme mutant, even though less dsRNA was made, leading to more profound effects on viral gene expression. Human Hap1 and A549 cells were genetically modified by CRISPR/Cas9 to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knock-out (KO) Hap1 cells in which RNase L is intrinsically inactive but only in PKR+RNase L double KO (DKO) A549 cells, indicating that both pathways decreased replication equivalently and no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than wild-type virus) in DKO A549 cells but not in DKO Hap1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but non-permissive for VACV; however wild-type and mutant viruses replicated in triple KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L were sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant particularly in HAP1 DKO cells indicated additional translational defects.

    IMPORTANCE Viruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded (ds) RNA triggers in host cells. We showed that the dsRNA produced in excess in cells infected with a vaccinia virus (VACV) decapping enzyme mutant and by wild-type virus co-localized with the viral E3 protein in cytoplasmic viral factories. Novel human cell lines defective in either or both protein kinase R and RNase L dsRNA effector pathways and/or the cellular 5rrsquo; exonuclease Xrn1 were prepared by CRISPR/Cas9 gene editing. Inactivation of both pathways was necessary and sufficient to allow full replication of the E3 mutant and reverse the defect cause by inactivation of Xrn1, whereas the decapping enzyme mutant still exhibited defects in gene expression. The study provided new insights into functions of the VACV proteins and the well-characterized panel of CRISPR/Cas9 modified human cell lines should have broad applicability for studying innate dsRNA pathways.

  • Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection [PublishAheadOfPrint]

  • The four Brome mosaic virus (BMV) RNAs are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidates each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of post-translational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1, but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. CryoEM reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation affected CP-RNA interaction to modulate interaction with the encapsidated RNA, the release of three of the BMV RNAs. CLIP-seq experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contained regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.

    Importance: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus with its three genomic RNAs encapsidated in separate virions. Through proteomic structural, and biochemical analyses this work shows that post-translational modifications, specifically phosphorylation, on the capsid protein regulates capsid-RNA interaction, the stability of the virions, and affected viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the regulation of chromatin packaging by nucleosomes.

  • Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses [PublishAheadOfPrint]

  • Hepadnaviruses (HBVs) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs that allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome Shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motif I, II and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topology, characterized by three transmembrane domains. Most striking was that the BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host co-divergence over millions of years and host species jumping.

    IMPORTANCE Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegill, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

  • CD4-induced antibodies promote association of the HIV-1 envelope glycoprotein with CD4-binding site antibodies [PublishAheadOfPrint]

  • The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1 neutralizing antibodies nndash; CD4 binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer, but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5.

    IMPORTANCE STATEMENT Potent HIV-1 neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes.

  • ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments [PublishAheadOfPrint]

  • Human rhinovirus (HRV) -A89 and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here we show, by immunofluorescence microscopy, that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant-negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab-11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating.

    IMPORTANCE Based on similarity of their RNA genomic sequences the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A and all HRV-B use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B respectively, but entering the cell via the same receptor, direct the endocytosis machinery to ferry them along distinct pathways towards different endocytic compartments for uncoating.

  • Analysis of the Cis and Trans Requirements for DNA Replication at the Right End Hairpin of the Human Bocavirus 1 Genome [PublishAheadOfPrint]

  • Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in Dependoparvovirus adeno-associated virus 2 (AAV2) and Protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of Bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney (HEK) 293 cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large non-structural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 are required for the NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small non-structural protein NP1 is required for HBoV1 DNA replication at OriR. The NP1 and other viral non-structural proteins (NS1-4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of the NP1 in viral DNA replication at OriR. Overall, our study revealed characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication.

    IMPORTANCE Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells, and produces progeny virions that are infectious in well-differentiated airway epithelial cells. Recombinant AAV2 vector pseudotyped with HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin to a 46-nt sequence in the right-end hairpin, which contains both NS1 nicking and binding sites. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin, and to design next generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

  • DC-SIGN and L-SIGN are attachment factors that promote infection of target cells by human metapneumovirus in the presence or absence of cellular glycosaminoglycans. [PublishAheadOfPrint]

  • It is well established that glycosaminoglcyans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating virions at the cell-surface to promote interaction with other receptors for virus entry and infection. There is increasing evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells, however definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the widespread expression of cell surface GAGs. pgsA745 Chinese Hamster Ovary (CHO) cells are deficient in expression of cell-surface GAGs and resistant to HMPV infection. Herein, we demonstrate that expression of the Ca2+-dependent C-type lectin receptors (CLRs) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection. Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin-dependent and inhibited by mannan, but not by pre-treatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming CLRs can promote HMPV infection in the presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type or endocytosis-defective mutants of DC/L-SIGN indicated that the endocytic function of CLRs was not essential, but could contribute to HMPV infection of GAG-deficient cells. Together, these studies confirm a role for CLR as attachment factors and entry receptors for HMPV infection. Moreover, they define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity to HMPV infection can be rescued following expression of a single cell-surface receptor.

    IMPORTANCE On the surface of CHO cells, glycosaminoglycans (GAGs) function as the major attachment factor for human metapneumoviruses (HMPV), promoting dynamin-independent infection. Consistent with this, GAG-deficient pgaA745 CHO cells are resistant to HMPV. However, expression of DC-SIGN or L-SIGN rendered pgsA745 cells permissive to dynamin-dependent infection by HMPV, although the endocytic function of DC/L-SIGN was not essential for, but could contribute to, enhanced infection. These studies provide direct evidence implicating DC/L-SIGN as alternate attachment factors for HMPV attachment, promoting dynamin-dependent infection via other unknown receptors in the absence of GAGs. Moreover, they describe a unique experimental system for the assessment of putative attachment and entry receptors for HMPV.

  • miR-155 is necessary for efficient gammaherpesvirus reactivation from latency, but not for latency establishment. [PublishAheadOfPrint]

  • MicroRNA-155 (miR-155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). There is in vitro evidence to support a critical role for cellular miR-155 and viral miR-155 homologues in the establishment of gammaherpesvirus latency in B cells. We sought to determine the contribution of miR-155 to the establishment and maintenance of latency in vivo using murine gammaherpesvirus (MHV-68) infection. MHV-68-infected mice deficient in miR-155 exhibited decreases in GC B cells and Tfh cells. However, the frequency of spleen cells harboring latent MHV-68 genomes was the same in both miR-155 deficient and WT mice. Similar latent loads were also observed in mixed bone marrow chimeric mice, where B cell-extrinsic effects of miR-155 deficiency were normalized. Interestingly, we observed markedly lower efficiency of reactivation from latency in miR-155 deficient cells, indicating an important role for miR-155 in this process. These in vivo data complement previous in vitro studies and reach the conclusion that miR-155 is not necessary for the establishment or maintenance of gammaherpesvirus latency, but does affect reactivation efficiency.

    IMPORTANCE: Gammaherpesvirus infection leads to severe disease in immunosuppressed populations. MiR-155 has been shown to play important roles in many pathological processes, including tumorigenesis and diseases caused by an overly aggressive immune response. Our work provides valuable in vivo data showing that miR-155 is dispensable for gammaherpesvirus latency, but it is critical for reactivation from latency, which is a crucial step in the viral life cycle.

  • Cell-free versus cell-to-cell infection by HIV-1 and HTLV-1: exploring the link among viral source, viral trafficking and viral replication [PublishAheadOfPrint]

  • HIV-1 and HTLV-1 are complex retroviruses mainly infecting CD4+ T lymphocytes. In addition, antigen-presenting cells such as dendritic cells (DCs) are targeted in vivo by both viruses, although to a lesser extend. Interaction of HIV-1 with DCs plays a key role in viral dissemination from the mucosa to CD4+ T lymphocytes present in lymphoid organs. While similar mechanisms may occur for HTLV-1 as well, most HTLV-1 data were obtained from T-cell studies, and little is known regarding the trafficking of this virus in DCs. We first compared the efficiency of cell-free versus cell-associated viral sources of both retroviruses at infecting DCs. We showed that both HIV-1 and HTLV-1 cell-free particles are poorly efficient at productively infecting DCs, except if DC-SIGN has been engaged. Furthermore, while SAMHD1 accounts for restriction of cell-free HIV-1 infection, it is not involved in HTLV-1 restriction. In addition, cell-free viruses mainly lead to a non-productive DC infection leading to trans-infection of T-cell a process important for HIV-1 spread but not for that of HTLV-1. Finally, we show that T:DC cell-to-cell transfer implies viral trafficking in vesicles that may both increase productive infection of DCs ("cis-infection") and allow viral escape from immune surveillance. Altogether, these observations allowed us to draw a model on HTLV-1 and HIV-1 trafficking in DCs.

  • Pestivirus Npro directly interacts with interferon regulatory factor 3 (IRF3) monomer and dimer [PublishAheadOfPrint]

  • Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I interferon (IFN-aalpha;/bbeta;) in response to viral infection. Upon viral infection, IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses such as classical swine fever virus (CSFV) target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein, Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We use recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins, and forms a soluble 1:1 complex. The full-length IRF3, but not merely either of the individual domains, is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator CREB-binding protein (CBP). The results indicate that the Npro binding site on IRF3 encompasses a region unperturbed by the phosphorylation and subsequent activation of IRF3, which include the dimer interface and CBP binding site.

    IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of Npro interaction with IRF3, including IRF3 species (inactive monomer vs. activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional co-factor, the CREB-binding protein. This is the first study to demonstrate that Npro is able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation.

  • A Novel Non-Replication Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection [PublishAheadOfPrint]

  • Congenital cytomegalovirus (CMV) is a leading cause of mental retardation and deafness in newborns. The guinea pig is the only small animal model for congenital CMV. A novel CMV vaccine was investigated as an intervention strategy against congenital guinea pig cytomegalovirus (GPCMV). In this disabled infectious single cycle (DISC) vaccine strategy, a GPCMV mutant lacked the ability to express an essential capsid gene (UL85 homolog, GP85), except when grown on a complementing cell line. In vaccinated animals, the GP85 mutant virus (GP85 DISC) induced an antibody response to important glycoprotein complexes considered neutralizing target antigens (gB, gH/gL/gO and gM/gN). The vaccine also generated a T cell response to the pp65 homolog (GP83), determined via a newly established guinea pig interferon gamma ELISPOT assay. In a congenital protection study, GP85 DISC vaccinated animals and non-vaccinated control group were challenged during pregnancy with wild type GPCMV (10^5 pfu). Animals went to term and viral load in target organs of pups analyzed. Based on live pup births in the vaccinated and control groups (94.1% vs 63.6%) the vaccine was successful in reducing mortality (P=0.0002). Additionally, pups from the vaccinated group had reduced CMV transmission with 23.5% infected target organs compared to 75.9% in the control group .Overall, these preliminary studies indicate that a DISC CMV vaccine strategy has the ability to induce an immune response similar to that of natural virus infection but has the increased safety of a non-replication competent virus, which makes this approach attractive as a CMV vaccine strategy.

    IMPORTANCE Congenital CMV is a leading cause of mental retardation and deafness in newborns. An effective vaccine against CMV still remains an elusive goal despite over fifty years of CMV research. The guinea pig, with a placenta structure similar to humans, is the only small animal model for congenital CMV and recapitulates disease symptoms (eg. deafness) in newborn pups. In this report, a novel vaccine strategy against congenital guinea pig cytomegalovirus (GPCMV) was developed, characterized and tested for efficacy. This disabled infectious single cycle (DISC) vaccine strategy induced a neutralizing antibody, or T cell response to important target antigens. In a congenital protection study, animals were protected against CMV in comparison to the non-vaccinated group (52% reduction of transmission). This novel vaccine was more effective than previously tested gB based vaccines and most other strategies live virus vaccines. Overall, the DISC vaccine is a safe and promising approach against congenital CMV.

  • Protein-protein interactions suggest novel activities of human cytomegalovirus tegument protein pUL103 [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, co-immunoprecipitation (co-IP) and BioID, to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, support the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expand our understanding of the multifunctional repertoire of pUL103; we detected HCMV pUL103 in nuclei of infected cells, and identified an ALIX-binding domain within the pUL103 sequence.

    IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein, pUL103, is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and cellular protein-protein interactions to identify and understand the range of pUL103 functions. We found that pUL103 interacts with cellular antiviral defense systems and proteins involved in organelle biogenesis and transport of cytoplasmic vesicles, and is present in infected cell nuclei. These results expand our understanding of the functional repertoire of pUL103 to include activities that extend from the earliest stages of infection through virion assembly and egress.

  • The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) augments viral lytic replication. [PublishAheadOfPrint]

  • The K1 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV lifecycle, we constructed a set of recombinant viruses that contained either wild-type K1, a deleted K1 ORF (KSHVK1), stop codons within the K1 ORF (KSHV-K15XSTOP) or a revertant K1 virus (KSHV-K1REV). We report that the KSHVK1 and KSHV-K15XSTOP recombinant viruses displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REV viruses upon reactivation from latency. Additionally, the KSHVK1 and KSHV-K15XSTOP recombinant viruses also yielded lower amounts of infectious progeny upon reactivation compared to WT KSHV and KSHV-K1REV virus infected cells. Upon reactivation from latency, WT KSHV and the KSHV-K1REV infected cells displayed activated Akt kinase as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV lifecycle.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the PI3K/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the lifecycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to WT virus and also yielded lower amounts of infectious progeny. We report that K1 plays an important role in the lifecycle of KSHV.

  • Characterization of a novel conformational GII.4 norovirus epitope: Implications in norovirus-host interactions [PublishAheadOfPrint]

  • Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver for their success. In order to study antibody recognition and evasion in detail, we have analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis and surface plasmon resonance. Our results show that the predicted epitope is composed of eleven amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) which is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes Q396R and N447D is recognized by the 3C3G3 monoclonal antibody, confirming the participation of these two sites in the epitope recognized by this antibody. Furthermore, a single change, Q396R, is able to modify the HBGA recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions both at the immunological, as well as at the receptor levels.

    IMPORTANCE Human noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be the most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein. The evolution of this epitope along sequential GII.4 variants has allowed noroviruses to evade previously elicited antibodies, thus explaining how the GII.4 genotype can persist over long periods, re-infecting the population. Our results also show that this epitope participates in the recognition of host receptors which have evolved over time as well.

  • Recapitulating Cross-Species Transmission of SIVcpz to Humans Using Humanized-BLT Mice [PublishAheadOfPrint]

  • The origins of HIV-1 have been widely accepted to be the consequence of simian immunodeficiency viruses from wild chimpanzees (SIVcpz) crossing over to humans. However, there has not been any in vivo study of SIVcpz infection of humans. Also, it remains largely unknown why only specific SIVcpz strains have achieved cross-species transmission and what transmission risk might exist for those SIVcpz strains that have not been found to infect humans. Closing this knowledge gap is essential for better understanding cross-species transmission and predicting the likelihood of additional cross-species transmissions of SIV into humans. Here we show hu-BLT mice are susceptible to all studied strains of SIVcpz, including the inferred ancestral viruses of pandemic and non-pandemic HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505), also strains that have not been found in humans (SIVcpzMT145 and SIVcpzBF1167). Importantly, the ability of SIVcpz to cross the interspecies barrier to infect humanized mice correlates with their phylogenetic distance to pandemic HIV-1. We also identified mutations of SIVcpzMB897 (Env G411R aamp; G413R) and SIVcpzBF1167 (Env H280Q aamp; Q380R) at 14 weeks post inoculation. Together, our results have recapitulated the events of SIVcpz cross-species transmission to humans and identified mutations that occurred during the first 16 weeks of infection, providing in vivo experimental evidence that the origins of HIV-1 are the consequence of SIVcpz crossing over to humans. This study also revealed that SIVcpz viruses whose inferred descendants have not been found in humans still have the potential to cause HIV-1 like zoonosis.

    IMPORTANCE It is believed that the origins of HIV-1 are the consequence of SIV viruses from wild chimpanzees crossing over to humans. However, the origins of HIV-1 have been linked back to only specific SIVcpz strains. There have been no experiments that directly test the in vivo cross-species transmissibility of SIVcpz strains to humans. This is the first in vivo study of SIVcpz cross-species transmission. With the humanized-BLT mouse model, we have provided in vivo experimental evidence of multiple SIVcpz strains crossing over to humans and identified several important mutations of divergent SIVcpz strains after long-term replication in human cells. We also found the cross-species transmission barrier of SIVcpz to humans correlates with their phylogenetic distance to pandemic HIV-1 group M. Importantly, this work provides evidence that SIVcpz viruses, whose inferred descendants have not been found in humans, still have the potential to cause a future HIV-1 like zoonotic outbreak.

  • LANA E3 Ubiquitin-Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation [PublishAheadOfPrint]

  • Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin-ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus-4 (MuHV-4) infection of mice as a model system to address the role of latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS-box (V199A, V199A/L202A or P203A/P206A) disrupted mLANA's ability to recruit ElonginC and Cullin5, thereby impairing the formation of the EC5SmLANA complex and mLANA's E3 ligase activity on host NF-B and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by EMSA, the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS-box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS-box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease.

    IMPORTANCE The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause life-long persistent infection and exert causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multi-copy episome, and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin-ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin-ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.

  • Macrophages in progressive HIV/SIV infections [PublishAheadOfPrint]

  • The cells that are targeted by primate lentiviruses (HIV and SIV) are of intense interest given the renewed effort to identify potential cures for HIV. These viruses have been reported to infect multiple cell lineages of hematopoietic origin including all phenotypic and functional CD4 T cell subsets. The two most commonly reported cell types that become infected in vivo are memory CD4 T cells and tissue-resident macrophages. Though viral infection of CD4 T cells is routinely detected in both HIV-infected humans and SIV-infected Asian macaques, significant viral infection of macrophages is only routinely observed in animal models wherein CD4 T cells are almost entirely depleted. Here we review the roles of macrophages in lentiviral disease progression, evidence that macrophages support viral replication in vivo, animal models where macrophage-mediated replication of SIV is thought to occur, how the virus can interact with macrophages in vivo, pathologies thought to be attributed to viral replication within macrophages, how viral replication in macrophages might contribute to the asymptomatic phase of HIV/SIV, and whether macrophages represent a long-lived reservoir for the virus.

  • Antiviral activity of myticin C peptide from mussel: an ancient defence against herpesviruses [PublishAheadOfPrint]

  • Little is known about the antiviral response in molluscs. As in other invertebrates, the interferon signalling pathways have not been identified, and in fact, there is a debate on whether invertebrates possess an antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, the interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naïve mussels confer resistance in oysters to Ostreid herpesvirus 1 (OsHV-1) when oyster haemocytes are incubated with mussel haemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel haemolymph and haemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel haemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible of the antiviral activity of mussel haemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting molluscs. Moreover, modified myticin C peptides showed antiviral activity against human herpes simplex viruses types 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications.

    IMPORTANCE Several bioactive molecules have been identified and isolated from marine invertebrates that have potential pharmaceutical or industrial applications. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel haemolymph and haemocytes, showed potential as an antiviral agent against Ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster farming sector. Both haemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster haemocytes. Additionally, a modified peptide derived from myticin C also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of molluscs.

  • Mitochondrial p53 contributes to reovirus-induced neuronal apoptosis and CNS injury in a mouse model of viral encephalitis [PublishAheadOfPrint]

  • The tumor suppressor p53 plays a critical part in determining cell fate both as a regulator of the transcription of several pro-apoptotic genes and through its binding interactions with Bcl-2 family proteins at the mitochondria. We now demonstrate that p53 protein levels increase in infected brains during reovirus encephalitis. This increase occurs in the cytoplasm of reovirus-infected neurons and is associated with the activation of caspase 3. Increased levels of p53 in reovirus-infected brains are not associated with increased expression of p53 mRNA suggesting that p53 regulation occurs at the protein level. Increased levels of p53 are also not associated with increased expression of p53-regulated, pro-apoptotic genes. In contrast, upregulated p53 accumulates in the mitochondria. Previous reports have demonstrated that the binding of p53 to Bak at the mitochondria causes Bak activation and results in apoptosis. We now show that Bak is activated, and that activated Bak is bound to p53 during reovirus encephalitis. In addition, survival is enhanced in reovirus infected Bak-/- mice compared to controls, demonstrating a role for Bak in reovirus pathogenesis. Inhibition of the mitochondrial translocation of p53with pifithrin mmu; prevents the formation of p53/Bak complexes following reovirus infection of ex vivo brain slice cultures and results in decreased apoptosis and tissue injury. These results suggest that the mitochondrial localization of p53 regulates reovirus-induced pathogensis in the CNS through its interactions with Bak.

    IMPORTANCE There are virtually no specific treatments of proven efficacy for virus-induced neuroinvasive diseases. A better understanding of the pathogenesis of virus-induced CNS injury is crucial for the rational development of novel therapies. Our studies demonstrate that p53 is activated in the brain following reovirus infection and may provide a therapeutic target for virus-induced CNS disease.

  • Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired During Fibroblast Passage Restores Pathogenesis in Immune Suppressed Guinea Pigs and in the Context of Congenital Infection [PublishAheadOfPrint]

  • Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Non-pathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping ORF of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in E. coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to N13R10. However, following experimental challenge of immune compromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 x 106-fold) viral loads in blood and end-organs compared to N13R10. In pregnant guinea pigs, challenge with doses gge;5 x 106 pfu of r129 virus resulted in levels of maternal viremia, congenital transmission, pup viral loads, IUGR, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129/GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC.

    Importance Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of HCMV UL128, UL130 and UL131A. These mutations can attenuate the course of infection when the virus when re-introduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frame-shift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

  • The capsid-CPSF6 interaction is dispensable for HIV-1 replication in primary cells but is selected during virus passage in vivo [PublishAheadOfPrint]

  • CPSF6, a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle including uncoating, nuclear entry and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+ T cells and humanized mice at a level comparable to the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains.

    IMPORTANCE CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identify a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known co-factors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.

  • Evidence that receptor destruction by the Sendai virus hemagglutinin-neuraminidase protein is responsible for homologous interference. [PublishAheadOfPrint]

  • Receptor destruction has been considered as one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus type 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach or penetrate the infected cells, and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that aalpha;2,3 linked sialic acids were specifically reduced in the SeV infected cells, but aalpha;2,6 linked sialic acids had not changed. As infection of IAV removed both aalpha;2,3 and aalpha;2,6 linked sialic acids, especially aalpha;2,3 linked sialic acids, IAV infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, aalpha;2,3 linked sialic acids is relevant to homologous interference by SeV.

    IMPORTANCE Viral interference is a classically observed phenomenon but the precise mechanism is not clear. Using SeV interference, we provided concrete evidence that reduction of aalpha;2,3 linked sialic acids receptor by the HN of SeV was closely related with viral interference. Since SeV infection resulted in decrease of only aalpha;2,3 linked sialic acids, IAV, which also utilized aalpha;2,6 linked sialic acids to initiate infection, superinfected the SeV infected cells. In contrast, SeV could not superinfect the IAV infected cells because both aalpha;2,3 and aalpha;2,6 linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

  • Changes in HIV-1 Capsid stability induced by common CTL-driven viral sequence mutations [PublishAheadOfPrint]

  • HIV-1-infected individuals encoding for protective HLA class I alleles exhibit better control of viremia and slower disease progression. Viral control in these individuals has been associated with strong and potent HIV-1-specific CTL responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL-escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes for the conical capsid core, and have been suggested to reduce viral replication capacity. In this study the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a Cyclosporine A (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted capsid uncoating half time. Interestingly, capsid stability was correlated to infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses.

    IMPORTANCE Sequence mutations within p24 Gag selected by CTL-responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here we demonstrate that dominant HLA-B27-associated CTL-escape mutations within HIV-1 capsid are leading to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.

  • Spatial-temporal patterns of viral amplification and interference initiated by a single infected cell [PublishAheadOfPrint]

  • When viruses infect their host cells they can make defective virus-like particles along with intact virus. Cells co-infected with virus and defective particles often exhibit interference of virus growth caused by the competition for resources by defective genomes. Recent reports of the co-existence and co-transmission in vivo of such defective interfering particles (DIPs), across epidemiological length and time scales, suggest a role in viral pathogenesis, but it is not known how DIPs impact infection spread, even under controlled culture conditions. Using fluorescence microscopy, we quantified co-infections of vesicular stomatitis virus (VSV) expressing a fluorescent reporter protein and its DIPs on BHK-21 host-cell monolayers. We found viral gene expression was more delayed, infections spread more slowly, and patterns of spread became more "patchy" for higher DIP inputs to the initial cell. To examine how infection spread might depend on the behavior of the initial co-infected cell we built a computational model, adapting a cellular automata (CA) approach to incorporate for the first time-kinetic data of virus growth. Specifically, changes in observed patterns of infection spread could be directly linked to previous high-throughput single-cell measures of virus-DIP co-infection. The CA model also provided testable hypotheses on the spatial-temporal distribution of the DIPs, which remain governed by their predator-prey interaction. More generally, this work offers a data-driven computational modeling approach to better understand how single infected cells impact the multi-round spread of virus infections across cell populations.

    IMPORTANCE Defective interfering particles (DIPs) compete with intact virus, depleting host-cell resources that are essential for virus growth and infection spread. However, it is not known how such competition, strong or weak, ultimately impacts how infections spread and cause disease. Here we address this unmet need by developing an integrated experimental-computational approach, which sheds new light on how infections spread. We anticipate our approach will also be useful in the development of DIPs as therapeutic agents to manage the spread of viral infections.

  • A role for the {alpha}V integrin subunit in Varicella Zoster Virus-mediated fusion and infection. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytia formation in VZV infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knockdown expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The aalpha;V integrin subunit was identified to mediate VZV gB/gH-gL fusion as its knockdown by shRNAs reduced fusion levels to 60% of control cells. A comparable reduction in fusion levels was observed when an anti-aalpha;V antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in aalpha;V knockdown cells infected with the VZV pOka strain relative to the control cells. This was demonstrated by reductions in plaque size, replication kinetics and virion entry in the aalpha;V subunit knockdown cells. Thus, the aalpha;V integrin subunit is important for VZV gB/gH-gL fusion and infection.

    IMPORTANCE Varicella Zoster Virus (VZV) is a highly infectious pathogen that causes chicken pox and shingles. A common complication of shingles is the excruciating painful condition called postherpetic neuralgia that has proven difficult to treat. While a vaccine is now available, it is not recommended for immune compromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins, gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.

  • Pandemic-seasonal H1N1 reassortants recovered from patient material display a phenotype similar to the seasonal parent. [PublishAheadOfPrint]

  • We have previously shown that eleven patients became naturally co-infected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during co-infection of host animals and in vitro, however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two co-infecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants when compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally co-infected patients.

    IMPORTANCE Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural co-infection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.

  • Structure and genome release mechanism of human cardiovirus Saffold virus-3 [PublishAheadOfPrint]

  • In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for non-enveloped viruses. We address this gap in our current knowledge by studying the uncoating of the non-enveloped human cardiovirus Saffold virus-3 (SAFV-3) of the family Picornaviridae. SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 AAring; by X-ray crystallography and an 11 AAring;-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N-termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral threefold and fivefold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many cardioviruses contain a disulfide bond formed by cysteins that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection.

    IMPORTANCE A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for non-enveloped viruses. We address this gap in our current knowledge by studying the genome release of human Saffold virus-3. Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus-3 particle expands and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.

  • Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified the E2 tryptophan 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown as these earlier studies predate the infectious HCV cell-culture (HCVcc) system. Here, we introduced four separate mutations (F, Y, A or R) at position 420 within the infectious JFH-1 HCVcc genome and characterized their effects on the viral cycle. Whilst all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with receptors SR-BI as well as CD81 were modulated which in-turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid-transfer activity, for virus entry. Furthermore, they were resistant to the drug erlotinib that targets EGFR (a host co-factor for HCV entry) and also blocks SR-BI dependent HDL-mediated enhancement of virus entry. Together, our data indicate a model where alteration at position 420 causes a subtle change in E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81 binding site in-turn favoring a particular internalization route. They further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.

    IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver disease causing up to 500000 deaths annually. The first step in the viral life-cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan 420 of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated the interaction with the SR-BI receptor and changes to these key interactions were found to affect the virus internalization route involving the host co-factor, EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor-point to stabilize the E2-CD81 interaction.

  • Virion structure of iflavirus slow bee paralysis virus at 2.6A resolution [PublishAheadOfPrint]

  • The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of non-enveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 AAring; and 2.6 AAring;. The overall structure of the virion resembles that of picornaviruses with the three major capsid proteins VP1-3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales. The protruding (P)-domains form "crowns" on the virion surface around each fivefold axis in one of the crystal forms. However, the P-domains are shifted 36 AAring; towards the threefold axis in the other crystal form. Furthermore, the P-domain contains the ser-his-asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV.

    IMPORTANCE Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding "crowns" on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry.

  • Arabidopsis RNA polymerases IV and V are required to establish H3K9 methylation, but not cytosine methylation, on geminivirus chromatin [PublishAheadOfPrint]

  • In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naïve loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation, but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2, and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin.

    IMPORTANCE In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to histone 3 lysine 9 dimethylation (H3K9me2). These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a re-evaluation of this view. We used Beet curly top virus (BCTV) to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naïve viral chromatin, and further that Pol IV-RdDM is responsible.

  • Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b. [PublishAheadOfPrint]

  • During infection of their host cell, viruses often inhibit production of host proteins, a process which is referred to as host shutoff. By doing this, viruses limit production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the Alpha- and Betacoronavirus genera, such as severe acute respiratory syndrome coronavirus (SARS-CoV) establish host shutoff via their non-structural protein 1 (nsp1). The genomes of Gamma- and Deltacoronaviruses however do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here we show that infectious bronchitis Gammacoronavirus (IBV) does induce host shutoff and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether we demonstrate that 5b is a functional equivalent of nsp1 thereby answering the long-standing question whether lack of nsp1 in Gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies.

    IMPORTANCE Many viruses inhibit protein synthesis of their host cell to enhance virus replication and antagonize anti-viral defense mechanisms. This process is referred to as llsquo;host-shutoff'. We have studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen, infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of the host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host.

  • Antiretroviral therapy in SIV-infected sooty mangabeys: implications for AIDS pathogenesis [PublishAheadOfPrint]

  • Simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) do not develop AIDS despite high levels of viremia. Key factors involved in the benign course of SIV infection in SMs are the absence of chronic immune activation and low levels of infection of CD4+ central memory (TCM) and stem cell memory (TSCM) T-cells. To better understand the role of virus replication in determining the main features of SIV infection in SMs, we treated twelve SMs with a potent antiretroviral therapy (ART) regimen for 2-12 months. We observed that ART suppressed viremia to llt;60 copies/ml of plasma in 10 out of 12 animals, and induced a variable decrease in the level of cell-associated SIV-DNA in peripheral blood (average fold-change: 0.9, 1.1, 1.5 and 3.7 for CD4+ transitional memory, TTM, TCM, effector memory, TEM, and TSCM, respectively). ART-treated SIV-infected SMs showed (i) increased percentage of circulating CD4+ TCM; (ii) increased levels of CD4+ T-cells in the rectal mucosa, and (iii) significant decline in the frequency of HLA-DR+CD8+ T-cells in blood and rectal mucosa. In addition, we observed that ART interruption resulted in rapid viral rebound in all SIV-infected SMs, indicating that the virus reservoir persists for at least a year under ART despite lower infection of CD4+ TCM and TSCM when compared to pathogenic SIV infection of macaques. Overall these data indicate that ART induces specific immunological changes in SIV-infected SMs, thus suggesting that virus replication impacts on the immune function even in the context of this clinically benign infection.

    IMPORTANCE Studies of natural, non-pathogenic Simian Immunodeficiency Virus (SIV) infection of African monkeys have provided important insights into the mechanisms responsible progression to AIDS during pathogenic HIV infection of humans and SIV infection of Asian macaques. In this study, we treated for the first time SIV-infected sooty mangabeys, a natural host for the infection, with a potent antiretroviral therapy (ART) regimen for a period ranging between 2 and 12 months and monitored in detail how suppression of virus replication impacts the main virological and immunological features of this non-pathogenic infection. The observed findings provide novel information on both the pathogenesis of residual immunological disease under ART during pathogenic infection and the mechanisms involved in virus persistence during primate lentiviral infections.

  • Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes [PublishAheadOfPrint]

  • African horse sickness virus, an orbivirus in the Reoviridae family, with nine different serotypes causes devastating disease in equids. The virion particle is composed of seven proteins, organized in three concentric layers, an outer made of VP2 and VP5, a VP7 middle layer and inner layer of VP3 that encloses a replicase complex of VP1, VP4 and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious vaccine candidates against all AHSV serotypes, taking into account not only immunogenic and safety properties, but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established highly efficient reverse genetics (RG) system for AHSV1 and subsequently, VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required exchange of two to five RNA segments to achieve equivalent titers of particles. In the VP6-complementary cells, all reassortant defective viruses could be amplified and propagated to high titers, but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor knock-out mice. Thus, these defective viruses have potential for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis and pathogenesis.

    IMPORTANCE African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, reaching up to 95% mortality in naïve horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through establishing a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes have been generated. These defective viruses achieved high titers in a VP6-complementing cell line, but failed to propagate in wild-type mammalian or insect cells. Importantly, these vaccine candidate strains showed strong protective efficacy against AHSV infection in an IFNARnndash;/nndash; mouse model.

  • Complex interplay between HIV-1 Capsid and MX2-independent IFN{alpha}-induced antiviral factors [PublishAheadOfPrint]

  • Type I interferons (IFNs), including IFNaalpha; upregulate an array of interferon-stimulated genes (ISGs) and potently suppress HIV-1 infectivity in CD4+ T cells, monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Recently, we and others identified the ISG myxovirus resistance 2 (MX2) to block HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection e.g. prior to (or during) reverse transcription remain to be defined. Here we show that HIV-1 CA mutants such as N74D or A105T, which both allow escape from inhibition by MX2 and the truncated version of the cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutant P90A, each exhibit increased sensitivity to IFNaalpha;-mediated inhibition. Using CRISPR/Cas9 technology, we demonstrate that the IFNaalpha;-hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant, but modestly increased the IFNaalpha;-sensitivity of wild type virus. Interestingly, the infectivity of wild type or P90A virus could be rescued from the MX2-independent IFNaalpha;-induced blocks in THP-1 cells by treatment with cyclosporine (Cs), or its non-immunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFNaalpha;-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shielding the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection.

    Importance Human immunodeficiency virus type-1 (HIV-1) is the causative of the acquired immunodeficiency syndrome (AIDS). During acute HIV-1 infection, numerous pro-inflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the post-entry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1 infected patients. The identities of the cellular antiviral factors, their viral targets and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early post-entry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival.

  • Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry [PublishAheadOfPrint]

  • ZMapp, a cocktail of three monoclonal antibodies (mAbs; c2G4, c4G7 and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these mAbs bind a soluble EBOV GP ectodomain, and that mAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7 and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viral-like particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, while c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three mAbs bind with high occupancy, and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+ endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19 kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is, therefore, by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza HA and HIV Env.

    IMPORTANCE The recent West African outbreak of ebolavirus caused the deaths of over 11,000 individuals. Hence there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three mAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography we provide structural information on how each of the mAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayedmmdash;embedded in the membrane and present at high densitymmdash;on filamentous viral-like particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the mAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically-primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

  • Co-infection with human CMV genetic variants in transplant recipients and its impact on anti-viral T cell immunity [PublishAheadOfPrint]

  • Reconstitution of T cell immunity is absolutely critical for the effective control of virus-associated infectious complications in hematopoietic stem cell transplant (HSCT) recipients. Co-infection with genetic variants of human cytomegalovirus (CMV) in transplant recipients has been linked to clinical disease manifestation, however how these genetic variants impact on T cell immune reconstitution remains poorly understood. Here we have evaluated dynamic changes in the emergence of genetic variants of CMV in HSCT recipients and correlated these changes with reconstitution of anti-viral T cell responses. Analysis of single nucleotide polymorphisms within sequences encoding HLA class I-restricted CMV epitopes from the immediate early 1 gene of CMV revealed that co-infection with genetically distinct variants of CMV was detected in 52% of patients. However in spite of exposure to multiple viral variants, the T cell responses in these patients were preferentially directed to a limited repertoire of HLA class I-restricted CMV epitopes, either conserved, variant or cross-reactive. More importantly, we also demonstrate that long-term control of CMV infection after HSCT is primarily mediated through the efficient induction of a stable anti-viral T cell immunity irrespective of the nature of the antigenic target. These observations provide important insights for the future design of anti-viral T cell-based immunotherapeutic strategies for transplant recipients emphasising the critical impact of robust immune reconstitution for efficient control of viral infection.

    IMPORTANCE Infection and disease caused by human Cytomegalovirus (CMV) remains a significant burden in patients undergoing haematopoietic stem cell transplantation (HSCT).The establishment of efficient immunological control, primarily mediated by cytotoxic T cells plays a critical role in preventing CMV-associated disease in transplant recipients. Recent evidence has also begun to investigate the impact genetic variation in CMV has upon disease outcome in transplant recipients. In this study we sought to investigate the role T cell immunity plays in recognising and controlling genetic variants of CMV. We demonstrate that while a significant proportion of HSCT recipients may be exposed to multiple genetic variants of CMV, this does not necessarily lead to immune control mediated via recognition of this genetic variation. Rather immune control is associated with the efficient establishment of a stable immune response predominantly directed against immunodominant conserved T cell epitopes.

  • RNA-binding of Ebola virus VP30 is essential for activating viral transcription [PublishAheadOfPrint]

  • The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral (-) RNA genome, which is complexed by the nucleoprotein, NP, VP35, the polymerase L, VP24, and VP30. While viral replication is exerted by the polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3rrsquo; -terminal 154 nucleotides of the EBOV genome as standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc-finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA-dependent, while formation of VP30 homohexamers and VP35 homotetramers are not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (-) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures.

    IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

  • P3N-PIPO, a Frameshift Product from P3, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas [PublishAheadOfPrint]

  • Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No. 30 and 90-1 (Cl-No.30 and Cl-90-1), but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reactionnndash;like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1. Cl-No.30 did not induce cell death but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that from Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV.

    IMPORTANCE Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than the non-breaking isolate Cl-No.30. Here, we show that a susceptible pea line recognized the increased P3N-PIPO amount produced by Cl-90-1 Br2 and activated the salicylic-acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in cyv1 pea and two susceptible peas. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

  • 1H NMR metabolomics of plasma unveils liver dysfunction in dengue patients [PublishAheadOfPrint]

  • Dengue, due to its global burden, is the most important arthropod-born flavivirus disease and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detecting changes in the metabolic profile associated with disease pathogenesis could help identifying markers of prognostic and diagnostic importance. We applied 1H NMR exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyzes to discriminate individuals with dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) presenting primary and secondary infection and subjects with non-specific infection (ND). Our results showed that a decrease in plasma LDL and VLDL discriminated dengue-infected from ND subjects and also those subjects with severe infection presented an even decrease in lipoproteins concentration when compared to subjects with mild infection. These results add to the ongoing discussion that manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine was characteristic of DENV infection and disease severity and an increase in plasma acetate discriminated subjects with DF and DHF from ND-subjects. Several other metabolites showed to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in plasma metabolome are suggestive of liver dysfunction and could provide insights into the underlying molecular mechanisms of dengue pathogenesis and could help discriminating individuals at risk to develop severe infection and to predict disease outcome.

    IMPORTANCE Dengue virus infection, due to its global burden, is the most important mosquito-born viral disease. There is no specific treatment for dengue disease and early detection lowers fatality rates to below 1%. In this study we observed the effects of dengue virus infection on the profile of small molecules in the blood of patients with mild and severe infection. Variations in the profile of these small molecules reflect the replication of dengue virus in different tissues and the extent of tissue damage during infection. The results of this study showed that the molecules that changed the most were VLDL and LDL lipoproteins and amino acids. We proposed that these changes reflect liver dysfunction and also that they can be used to discriminate subjects with mild and severe dengue infection.

  • Homology-based identification of a mutation in the coronavirus RNA-dependent RNA polymerase that confers resistance to multiple mutagens [PublishAheadOfPrint]

  • Positive-sense (+) RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than 10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it with solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. These results also indicate that, with regards to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multi-protein replicase/proofreading complex.

    IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity, that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other unrelated RNA viral polymerases, and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

  • A recombinant respiratory syncytial virus vaccine candidate attenuated by a low-fusion F protein is immunogenic and protective against challenge in cotton rats [PublishAheadOfPrint]

  • Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naïve infants. However, designing an LAV with appropriate attenuation, yet sufficient immunogenicity has proven challenging. Here, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for non-structural proteins NS1 and NS2 (dNS); deletion of the small hydrophobic protein (SH) gene; and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate RSV-A2-dNS-SH-BAF named "DB1" was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g tissue respectively compared to unvaccinated animals (P llt; 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity.

    IMPORTANCE RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naïve infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. Here, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized non-structural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate "DB1" was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

  • Identification and Characterization of Influenza Virus Entry Inhibitors Through Dual-Myxovirus High-Throughput Screening [PublishAheadOfPrint]

  • Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual-myxovirus high-throughput screening protocol, that combines a fully replication-competent IAV-WSN and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. Here, this protocol was applied to a screening campaign, assessing a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an insolate of the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hits to HA. Resistance profiling revealed two distinct escape mechanisms: primary-type resistance associated with reduced compound binding and secondary resistance with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose of the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path towards structure-aided lead development.

    Importance This study is one of the first to apply a fully replication-competent third generation IAV reporter strain to a large-scale HTS drug discovery campaign, allowing multi-cycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding into a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

  • HSV capsid localization to ESCRT-VPS4 complexes in the presence and absence of the large tegument protein UL36p. [PublishAheadOfPrint]

  • UL36p (VP1/2) is the largest protein encoded by HSV-1 and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A GFP-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT-envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association, but the remaining UL36p-null capsids were still able to engage with the ESCRT envelopment apparatus. It appears that, although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement in envelopment.

    Importance Envelopment of the HSV capsid is essential for the assembly of an infectious virion, and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, also known to be required for envelopment.

  • Two Polypyrimidine Tracts in Intron 4 of the Major Immediate-Early (MIE) Gene Are Critical for the Gene Expression Switching from IE1 to IE2 and for Replication of Human Cytomegalovirus [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) major immediate-early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), first 3 exons and first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's 4th intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a ChIP assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target.

    Importance CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than 2 proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified 2 Pys in intron 4 and found that the first Py is important and the second is required for the splicing and expression of IE2. We further investigated the mechanisms of gene switching from IE1 to IE2 and found that the 2 Pys are responsible for U2AF65rrsquo; binding with intron 4. Therefore, the Pys in intron 4 are the cis-elements that determine the fate of IE2 splicing. Furthermore, we found that a small RNA that is complementary to the intron 4 repressed IE2 expression. Hence, we provide the first piece of evidence for a unique mechanism of MIE gene regulation at the splicing level.

  • Phosphorylation of human metapneumovirus M2-1 protein upregulates viral replication and pathogenesis [PublishAheadOfPrint]

  • Human metapneumovirus (hMPV) is a major causative agent of upper and lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1 that plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using reverse genetics, recombinant hMPVs lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation sites in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wildtype hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication and pathogenesis in vivo.

    Importance The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional anti-termination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis in vivo. Recombinant hMPVs lacking phosphorylation sites in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines, as well as antiviral drugs for pneumoviruses.

  • Time-dependent rate phenomenon in viruses [PublishAheadOfPrint]

  • Among the most fundamental questions in viral evolutionary biology are how fast viruses evolve and how their rates vary among viruses and fluctuate through time. Traditionally, viruses are loosely classed into two groups: slow-evolving DNA viruses and fast-evolving RNA viruses. As viral evolutionary rate estimates become more available, it appears that the rates are negatively correlated with the measurement timescales, and that the boundary between the rates of DNA and RNA viruses might not be as clear as previously thought. In this study, we collected 396 viral evolutionary rate estimates across almost all viral genome types and replication strategies, and examined their rate dynamics. We showed that the time-dependent rate phenomenon exists across multiple levels of viral taxonomy, from the Baltimore classification viral groups to genera. We also showed that, by taking the rate-decay dynamics into account, a clear division between the rates of DNA and RNA as well as reverse-transcribing viruses could be recovered. Surprisingly, despite large differences in their biology, our analyses suggested that the rate-decay speed is independent of viral types, and thus it might be useful for better estimation of the evolutionary timescale of any virus. To illustrate this, we used our model to re-estimate evolutionary timescales of extant lentiviruses, which were previously suggested to be very young by standard phylogenetic analyses. Our analyses suggested that they are millions of years old, consistent with paleovirological evidence, and therefore for the first time, reconciled molecular analyses of ancient and extant viruses.

    IMPORTANCE This work provides direct evidence that viral evolutionary rate estimates decay with their measurement timescales, and that the rate-decay speeds do not differ significantly among viruses despite the vast differences in their molecular features. With the rate-decay dynamics adjusted for, the division between the rates of dsDNA, ssRNA, and ssDNA/reverse-transcribing viruses could be seen more clearly than before. Our results provide a guideline for further improvement of molecular clock. As a demonstration of this, we used our model to re-estimate the timescales of modern lentiviruses, which were previously thought to be very young, to be millions of years old. This result matches the estimate from paleovirological analyses, thus bridging the gap between ancient and extant viral evolutionary studies.

  • Suppression of IRG-1 improves immune lung injury after RSV infection by reducing ROS production [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infection is a common cause of lower respiratory tract illness in infants and children. RSV is a negative sense, single strand RNA (ssRNA) virus that mainly infects airway epithelial cells. Accumulating evidence indicates that reactive oxygen species (ROS) production is a major factor for pulmonary inflammation and tissue damage of RSV disease. We investigated immune responsive gene-1 (IRG1) expression during RSV infection since IRG1 has been shown to mediate innate immune response to intracellular bacterial pathogens by modulating ROS and itaconic acid production. We found that RSV infection induced IRG1expression in human A549 cells and in the lung tissues of RSV-infected mice. RSV infection or IRG1 overexpression promoted ROS production. Accordingly, knockdown of IRG1 induction blocked RSV-induced ROS production and proinflammatory cytokine gene expression. Finally, we showed that suppression of IRG1 induction reduced immune cell infiltration and prevented lung injury in RSV-infected mice. These results therefore link IRG1 induction to ROS production and immune lung injury after RSV infection.

    Importance Respiratory syncytial virus (RSV) infection is among the most common cause of childhood diseases. Recent studies identify ROS production as a contributing factor to RSV disease. We investigated the cause of ROS production and identified IRG1 as a critical factor linking ROS production to immune lung injury after RSV infection. We found that IRG1 was induced in A549 alveolar epithelial cells and in mouse lungs after RSV infection. Importantly, suppression of IRG1 induction reduced inflammatory cell infiltration and lung injury in mice. This study links IRG1 induction to oxidative damage and RSV disease. It also uncovers a potential therapeutic target in reducing RSV-caused lung injury.

  • Mutagenesis of S-adenosyl-L-methionine-binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity [PublishAheadOfPrint]

  • Eukaryotic mRNAs possess a methylated 5rrsquo; -guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5rrsquo; caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved S-adenosyl-L-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity in vitro. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon, and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to WT MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with interferon-bbeta;, and nsp14 G332A genomes were translated less efficiently both in vitro and during viral infection. In contrast, alanine substitution of MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by interferon-bbeta; compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ in vitro and during virus infection.

    IMPORTANCE Human coronaviruses, most notably SARS-CoV and MERS-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that alanine substitution of G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.

  • Human T-Lymphotropic Virus (HTLV)-1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier [PublishAheadOfPrint]

  • Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this report, we study the role of Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically-infected cell lines and primary infected CD4+ T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-B pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients shows that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis.

    Importance Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.

  • Mutants at the 2-fold interface of AAV2 structural proteins suggest a role in viral transcription for AAV capsids [PublishAheadOfPrint]

  • We previously reported that an amino acid substitution, Y704A, near the 2 fold interface of AAV was defective for transcription of the packaged genome (Salganik et al, 2014, J Virol, 88:1071). In this report we have characterized the defect in 6 additional capsid mutants located in a region approximately 30AAring; in diameter on the surface of the AAV2 capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3-6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA post infection. In addition, wild type capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody bound wild type capsids, were able to enter the cell, travel to the nucleus, uncoat and synthesize a second strand, but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2 fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome it delivers to the nucleus.

    IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes post infection, e.g., retroviruses. A number of non-enveloped viruses, including AAV, carry protease that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of a non-enveloped viral capsid that appears to have a role in promoting transcription. A total of six mutants at the AAV capsid 2 fold interface were shown to have a severe defect in expressing their genomes, and the defect was at the level of mRNA accumulation. This suggests that AAV capsids have a novel role in promoting transcription of the genomes they have packaged. Since wt virions could not complement the mutant viruses, and the mutant viruses did not effectively inhibit wt gene expression, our results suggest that the capsid exerts its effect on transcription in cis.

  • Vaccine-elicited mucosal and systemic antibody responses are associated with reduced simian immunodeficiency viremia in infant rhesus macaques [PublishAheadOfPrint]

  • Despite significant progress in reducing peri-partum mother-to-child transmission (MTCT) of HIV with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor, and breast milk exposure to HIV accounts for up to 44% of MTCT. As abstinence from breastfeeding is not recommended, alternative means are needed to prevent MTCT of HIV. We have previously shown that oral vaccination at birth with live attenuated Mycobacterium tuberculosis (Mtb) strains expressing SIV genes safely induces persistent SIV-specific cellular and humoral immune responses both systemically and at the oral and intestinal mucosa. Here we tested the ability of oral Mtb vaccine strains expressing SIV Env and Gag proteins, followed by systemic heterologous (MVA-SIV Env/Gag/Pol) boosting, to protect neonatal macaques against oral SIV challenge. While vaccination did not protect infant macaques against oral SIV acquisition, a subset of immunized animals had significantly lower peak viremia which inversely correlated with pre-challenge SIV Env-specific salivary and intestinal IgA responses and higher avidity SIV Env-specific IgG in plasma. These controller animals also maintained CD4+ T cell populations better and showed reduced tissue pathology compared to non-controller animals. We show that infants vaccinated at birth can develop vaccine-induced SIV specific IgA and IgG antibodies and cellular immune responses within weeks of life. Our data further suggest that affinity maturation of vaccine-induced plasma antibodies and induction of mucosal IgA responses at potential SIV entry sites are associated with better control of viral replication, thereby likely reducing SIV morbidity.

    IMPORTANCE Despite significant progress in reducing peri-partum mother-to-child transmission (MTCT) of HIV with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor. Breast milk exposure to HIV accounts for up to 44% of MTCT. Alternative measures, in addition to ART, are needed to achieve the goal of an AIDS-free generation. Pediatric HIV vaccines constitute a core component of such efforts.

    The results of our pediatric vaccine study highlight the potential importance of vaccine-elicited mucosal Env-specific IgA responses in combination with high-avidity systemic Env-specific IgG in protection against oral SIV transmission and control of viral replication in infant macaques. The induction of potent mucosal IgA antibodies by our vaccine is remarkable considering the age-dependent development of mucosal IgA responses post-birth. A deeper understanding of postnatal immune development may inform the design of improved vaccine strategies to enhance systemic and mucosal SIV/HIV antibody responses.

  • Ebola virus does not induce stress granule formation during infection and sequesters stress granule proteins within viral inclusions [PublishAheadOfPrint]

  • A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the translation initiation factor eIF2aalpha;. Here, we show that selected SG proteins are sequestered within EBOV inclusions where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2aalpha; phosphorylation at any time post infection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions.

    Importance Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

  • Using the E4orf6-based E3 ubiquitin ligase as a tool to analyze the evolution of adenoviruses [PublishAheadOfPrint]

  • E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information we set out to determine if other adenoviruses also possess the ability to form the ligase complex, and if so to predict Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage in E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly in non-human primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. We also suggest based on analysis of Cullin selection that the majority of human adenoviruses, which exhibit a broader tropism for the eye and respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastro-intestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that contain a conserved xCxC motif followed by, in most cases, a BC-box motif.

    Importance Two early adenoviral proteins, E4orf6 and E1B55K form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates leading to their degradation by the proteasome. In a study with representatives of each human adenovirus species we (and others) have previously discovered that some viruses use Cul2 to form the complex while others use Cul5. In the present study we have expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses contain the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of species Human mastadenovirus A, F and G. We have also identified previously unrecognized E4orf6 genes in the aviadenoviruses that contain motifs permitting formation of the ubiquitin ligase.

  • Nonstructural 3 protein of hepatitis C virus modulates tribbles homolog 3/Akt signaling pathway for persistent viral infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By RNA-Seq analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 is initially identified as a binding partner of protein kinase B (PKB, also known as Akt). TRIB3 blocks phosphorylation of Akt and induces apoptosis under ER stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increase of RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, TRIB3 and Akt association was disrupted by HCV NS3 and thus TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote ERK phosphorylation, AP-1 activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis.

    IMPORTANCE TRIB3 is a pseudokinase protein which acts as an adaptor in signaling pathway for important cellular processes. So far, functional involvement of TRIB3 in virus-infected cells has not been demonstrated yet. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels and thus overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates TRIB3-Akt signaling pathway to establish persistent viral infection.

  • Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation. [PublishAheadOfPrint]

  • Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV) replicates via an RNA intermediate and is error-prone, leading to rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome, generated via splicing (spHBV). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. SpHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from longitudinal samples of a post-liver transplant CHB subject, collected over a 15-year period that included the liver explant. By employing novel bioinformatics methods, this analysis showed a complex dynamics of the viral population across a period of changing treatment regimens. The spHBV detected in the liver explant remained present post-transplantation, along with emergence of a highly diverse novel spHBV population as well as variants with multiple deletions in the preS genes. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occur with known drug resistant mutations, highlight the relevance of using full genome deep sequencing and support the hypothesis that drug resistance involves interactions across the full-length HBV genome.

    IMPORTANCE Single-molecule sequencing allowed characterising, in unprecedented detail, the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimes. This analysis also showed rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open source bioinformatics tools are freely available, with the capacity to easily identify potential spliced variants from deep sequencing data.

  • Dengue virus subverts host innate immunity by targeting adaptor protein MAVS [PublishAheadOfPrint]

  • Dengue virus (DENV) is the most common mosquito-borne virus infecting humans and is currently a serious global health challenge. To establish infection in its host cells, DENV must subvert the production and/or antiviral effects of interferon (IFN). The aim of this study was to understand the mechanisms by which DENV suppresses IFN production. We determined that DENV NS4A interacts with mitochondrial antiviral signaling protein (MAVS), which has previously been found to activate NF-B and IFN regulatory factor 3 (IRF3), thus inducing type I IFN in the mitochondria-associated endoplasmic reticulum membranes (MAM). We further demonstrated that NS4A is associated with the N-terminal CARD-like domain (CL) and the C-terminal transmembrane domain (TM) of MAVS. This association prevented the binding of MAVS to RIG-I, resulting in repression of RIG-I-induced IRF3 activation and, consequently, abrogation of IFN production. Collectively, our findings illustrate a new molecular mechanism by which DENV evades the host immune system and suggest new targets for anti-DENV strategies.

    IMPORTANCE Type I interferon (IFN) constitutes the first line of host defense against invading viruses. To successfully establish infection, dengue virus (DENV) must counteract either the production or the function of IFN. The mechanism by which DENV suppresses IFN production is poorly understood and characterized. In this study, we demonstrate that the DENV NS4A protein plays an important role in suppressing interferon production through binding MAVS and disrupting the RIG-I-MAVS interaction in the mitochondria-associated endoplasmic reticulum membranes (MAM). Our study reveals MAVS as a novel host target of NS4A and provides a molecular mechanism for DENV's evasion of the host innate immune response. These findings have important implications for understanding the pathogenesis of DENV and may provide new insights into using NS4A as a therapeutic and/or prevention target.

  • Interaction Between Non-Structural Proteins NS4B And NS5A Is Essential For Proper NS5A Localization And Hepatitis C Virus RNA Replication [PublishAheadOfPrint]

  • The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in plane into the outer ER derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy we identified a number of essential residues for replication on the charged face of NS5A anchor, and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant, and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions.

    IMPORTANCE The information about the functional role of cytosolic face of NS5A anchoring helix remains obscure. In this study we show that, while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays key role during HCV replication by mediating interaction of NS5A with other HCV nonstructural proteins via NS4B. Such interaction determines the subcellular localization of NS5A by engaging NS5A into HCV replication process during the formation of functional HCV replication complex. Thus, collectively it can be stated, that the current study findings provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide the platform to gain more insights about the molecular architecture of HCV replication complexes.

  • Tegument protein ORF45 plays an essential role in virion morphogenesis of murine gammaherpesvirus-68 [PublishAheadOfPrint]

  • Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses, however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus-68 (MHV-68) ORF45 and found that it was incorporated into virions around the site of host-derived vesicles. Notably, absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.

  • Critical role of the PA-X C-terminal domain of influenza A virus on its subcellular localization and shutoff activity [PublishAheadOfPrint]

  • PA-X is a recently identified influenza virus protein, which is composed of PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain. However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region on shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.

    IMPORTANCE Influenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

  • Release of infectious hepatitis C virus from Huh7 cells occurs via a trans-Golgi network to endosome pathway independent of VLDL [PublishAheadOfPrint]

  • The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosome sorting complex required for transport (ESCRT). Here, we show a critical role of TGN-endosome trafficking during the assembly, but principally the secretion of infectious virus. This was demonstrated by both siRNA mediated silencing of TGN-associated adaptor proteins, and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of either triglyceride, ApoB or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct to that of VLDL. Finally we show that HCV NS2 perturbs trans-Golgi network (TGN) architecture, redistributing TGN membranes to closely associate with HCV core residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, whist essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins.

    Importance. The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects an unique modification to cellular trafficking pathways.

  • HIV-1-Specific Chimeric Antigen Receptors Based on Broadly-Neutralizing Antibodies [PublishAheadOfPrint]

  • Although the use chimeric antigen receptors (CARs) based on single chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain, and was abandoned for lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (bnAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined bnAbs varying in binding sites and generated single chain antibody-based CARs. These included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8+ T lymphocytes. Their antiviral activity was reproducible but varied according to the strain of virus. These findings indicated that bnAbs are excellent candidates for developing novel CARs to consider in the immunotherapeutic treatment of HIV-1.

    IMPORTANCE While chimeric antigen receptors (CARs) using single chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were for HIV-1 infection. However, those trials failed and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on using CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (bnAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse bnAb types, and show that all are functional against HIV-1.

  • Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids, and interacts with the components of the nuclear egress complex (NEC), namely pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 may serve as an important link between nucleocapsid maturation and nuclear egress.

    IMPORTANCE Human cytomegalovirus (HCMV) causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point towards an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

  • MAVS expressed by hematopoietic cells is critical for control of West Nile virus infection and pathogenesis [PublishAheadOfPrint]

  • West Nile virus (WNV) is the most important cause of epidemic encephalitis in North America. Innate immune responses, which are critical for control of WNV infection, are initiated by signaling through pathogen recognition receptors, RIG-I and MDA5, and their downstream adaptor molecule, MAVS. Here we show that a deficiency of MAVS in hematopoietic cells resulted in increased mortality and delayed WNV clearance from the brain. In Mavs-/- mice, a dysregulated immune response was detected, characterized by a massive influx of macrophages and virus-specific T cells into the infected brain. These T cells were polyfunctional and lysed peptide-pulsed target cells in vitro. However, virus-specific T cells in the brains of infected Mavs-/- mice exhibited lower functional avidity than those in wild-type animals, and even virus-specific memory T cells generated by prior immunization could not protect Mavs-/- mice from WNV-induced lethal disease. Concomitant with ineffective virus clearance, macrophage numbers were increased in the Mavs-/- brain and both macrophages and microglia exhibited an activated phenotype. Microarray analyses of leukocytes in the infected Mavs-/- brain showed a preferential expression of genes associated with activation and inflammation. Together, these results demonstrate a critical role for MAVS in hematopoietic cells in augmenting the kinetics of WNV clearance and thereby preventing a dysregulated and pathogenic immune response.

    IMPORTANCE West Nile virus (WNV) is the most important cause of mosquito-transmitted encephalitis in the United States. The innate immune response is known to be critical for protection in infected mice. Here, we show that expression of MAVS, a key adaptor molecule in the RIG-I-like receptor RNA sensing pathway, in hematopoietic cells is critical for protection from lethal WNV infection. In the absence of MAVS, there is a massive infiltration of myeloid cells and virus-specific T cells into the brain and over-exuberant production of pro-inflammatory cytokines. These results demonstrate the important role that MAVS expression in hematopoietic cells has in regulating the inflammatory response in the WNV-infected brain.

  • Viral evasion and manipulation of host RNA quality control pathways [PublishAheadOfPrint]

  • Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping, open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This review focuses on ways in which viral RNAs run afoul of cellular the mRNA quality control and decay machinery, as well as strategies developed by viruses to circumvent or exploit cellular mRNA surveillance.

  • Glutathione depletion is linked with Th2 polarization in mice with a retrovirus-induced immunodeficiency syndrome, MAIDS: role of pro-glutathione molecules as immunotherapeutics [PublishAheadOfPrint]

  • LP-BM5 murine leukemia virus injection causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting in GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels was measured in pancreas and brain respectively at 5 weeks post infection. Th2 immune response was predominant at all the times investigated as revealed by the expression of Th1/Th2 cytokines. Furthermore, an investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of iNOS, a marker of classical activation of macrophages, was detected only when Th1 cytokines were more expressed. In vitro studies revealed that during the very early phases of infection, GSH depletion and down-regulation of interleukin-12 p40 mRNA were correlated with the level of the infecting dose of LP-BM5 used to infect macrophages. Treatment of LP-BM5-infected mice with I-152, a N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased IFN- production while decreased Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection, and I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice.

    IMPORTANCE This study represents the first report of association between Th2 polarization and alteration of the redox state in LP-BM5 infection. Moreover, it is shown the evidence that LP-BM5 infection causes a decrease in thiol content of peritoneal macrophages which can influence IL-12 production.

    The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection re-establishing the Th1/Th2 balance. The immunotherapy based on the use of pro-GSH molecules would permit to more effectively combat LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and Th1/Th2 imbalance.

  • Generating bona fide mammalian prions with internal deletions. [PublishAheadOfPrint]

  • Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha helical content, loosing the three alpha helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C-terminus of ovine PrP, as this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naïve homologous and wild-type PrP expressing cells. Even more, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.

    IMPORTANCE Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date resolution of prion structure remains elusive. Thus to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C-terminus of alpha-helix H2, did not hamper prion formation, but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

  • A Genome-Wide RNAi Screen Identifies a Role for Wnt/Beta-Catenin Signaling During Rift Valley Fever Virus Infection [PublishAheadOfPrint]

  • Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was pre-activated, was reduced with knockdown of bbeta;-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed anti-viral therapeutics.

    IMPORTANCE: RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g. Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully-licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

  • Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium Between X4 and R5 Viruses In the V1/V2 and V3 Variable Regions [PublishAheadOfPrint]

  • HIV-1 requires the CD4 receptor and a co-receptor (CCR5 (R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Co-receptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late stage and 6 early stage subjects. We also used end-point dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false positive rate [FPR] lle; 2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), significantly higher than has been seen previously using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with intermediate FPR (2nndash;20%) could be either X4/dual or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of less than 10%, and these carried a distinctive sequence change. Phylogenetic analysis of both V1/V2 and C2/V3 regions showed evidence of recombination within but very little between the X4 and R5 lineages, suggesting these populations are genetically isolated.

    IMPORTANCE Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed using next generation sequencing (NGS) and with greater depth of sampling than SGA. Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting X4 and R5 variants are genetically isolated and may be replicating in different cell types, or that X4/R5 recombinants have reduced fitness.

  • A role of influenza exposure history in determining pandemic susceptibility and CD8+ T cell responses [PublishAheadOfPrint]

  • Novel influenza viruses often cause differential infection patterns across different age groups, defined as heterogeneous demographic susceptibility. This occurred during the A/H2N2 pandemic, when children experienced higher influenza attack rates than adults. As the recognition of conserved epitopes across influenza subtypes by CD8+ cytotoxic T lymphocytes (CTLs) limit influenza disease, we hypothesised that conservation of CTL antigenic peptides (Ag-p) in viruses circulating before the pH2N2-1957 may have resulted in differential CTL immunity. We compared viruses isolated in the years preceding the pandemic (1941nndash;1957) to which children and adults were exposed, with viruses circulating decades earlier (1918nndash;1940), which could infect adults only. Consistent with phylogenetic models, influenza viruses circulating 1941-1957, which infected children, shared with pH2N2 the majority (~89%) of CTL-peptides within the most immunogenic nucleoprotein, matrix-1 and polymerase basic-1, thus providing evidence for minimal pH2N2 CTL escape in children. Our study, however, identified potential CTL immune evasion from pH2N2 irrespective of age, within HLA-A*03:01+ individuals for PB1471-L473V/N476I variants and HLA-B*15:01+ population for NP404-414-V408I mutant. Further experiments using the murine model of B-cell-deficient mice showed that multiple influenza infections resulted in superior protection from influenza-induced morbidity, coinciding with accumulation of tissue resident memory CD8+ T-cells in the lung. Our study suggests that protection against H2N2-1957 pandemic influenza was most likely linked to the number of influenza virus infections prior to the pandemic challenge rather than differential pre-existing CTL immunity. Thus, the regimen of a CTL-based vaccine/vaccine-component may benefit from periodic boosting to achieve fully protective, asymptomatic influenza infection.

    IMPORTANCE Due to a lack of cross-reactive neutralizing antibodies, children are particularly susceptible to influenza infections caused by novel viral strains. Pre-existing T cell immunity directed at conserved viral regions, however, can provide protection against influenza viruses, promote rapid recovery and better clinical outcomes. When we asked whether high susceptibility of children (as compared to adults) to the pandemic H2N2 influenza strain was associated with immune evasion from T-cell immunity, we found high conservation within T-cell antigenic regions in pandemic H2N2. However, the number of influenza infections prior to the challenge was linked to protective, asymptomatic infections and establishment of tissue resident memory T cells. Our study supports development of vaccines that prime and boost T cells to elicit cross-strain protective T cells, especially tissue resident memory T cells, for life-long immunity against distinct influenza viruses.

  • MF59 adjuvant effects on inducing isotype-switched IgG antibodies and protection after immunization with T-dependent influenza virus vaccine in the absence of CD4+ T cells [PublishAheadOfPrint]

  • CD4+ T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4+ T cells in the adjuvant effects, we investigated the efficacy of T-dependent influenza virus split vaccine with MF59rreg; or alum in CD4-knockout (CD4KO) and wild-type (WT) mice. CD4+ T cells were required for the induction of IgG antibody responses to split vaccine and alum adjuvant effects. In contrast, MF59rreg; was found to be highly effective in raising isotype-switched IgG antibodies to T-dependent influenza split vaccine in CD4KO mice or CD4-depleted WT mice, equivalent to those in intact WT mice, thus overcoming the deficiency of CD4+ T cells in helping B cells and inducing immunity against influenza virus. MF59-adjuvanted influenza split vaccination was able to induce protective CD8+ T cells and long-lived antibody-secreting cells in CD4KO mice. MF59 adjuvant effects in CD4KO mice might be associated with uric acid, inflammatory cytokines, and recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, elderly, and immune-compromised patients as well as in healthy adults.

    IMPORTANCE MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4+ T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to T-dependent influenza vaccine independent of CD4+ T cells. These findings are highly significant (1). MF59 can overcome a defect of CD4+ T cells in inducing protective immunity to T-dependent influenza split vaccination (2). A CD4-independent pathway can be an alternative mechanism for certain adjuvants such as MF59 (3). This study has significant implications for improving vaccine efficacies in young children, elderly, and immune-compromised populations.

  • Notwithstanding circumstantial alibis, cytotoxic T cells can be major killers of HIV-1 infected cells. [PublishAheadOfPrint]

  • Several experiments suggest that in the chronic phase of HIV-1 infection CD8+cytotoxic T lymphocytes (CTL) contribute very little to the death of productively infected cells. First, the expected life span of productively infected cells is fairly long, i.e., about one day. Second, this life span is hardly affected by the depletion of CD8+T cells. Third, the rate at which mutants escaping a CTL response take over the viral population tends to be slow. Our main result is that all these observations are perfectly compatible with killing rates that are much faster than one per day once we invoke the fact that infected cells proceed through an eclipse phase of about one day before they start producing virus. Assuming that the major protective effect of CTL is cytolytic, we demonstrate that mathematical models with an eclipse phase account for the data when the killing is fast, and when it varies over the life cycle of infected cells. Considering the steady state corresponding to the chronic phase of the infection, we find that the rate of immune escape, and the rate at which the viral load increases following CD8+T cell depletion, should reflect the viral replication rate, . A meta analysis of previous data shows that viral replication rates during chronic infection vary between 0.5lle;lle;1 day-1. Balancing such fast viral replication requires killing rates that are several times larger than , implying that most productively infected cells would die by cytolytic effects.

    Importance Most current data suggest that Cytotoxic T cells (CTL) mediate their control of HIV-1 infection by non-lytic mechanisms, i.e., data suggest that CTL hardly kill. This interpretation of these data has been based upon the general mathematical model for HIV infection. Because this model ignores the eclipse phase between the infection of a target cell and the start of viral production by that cell, we re-analyze the same data sets with novel models that do account for the eclipse phase. We find that the data are perfectly consistent with lytic control by CTL, and predict that most productively infected cells are killed by CTL. Because that the killing rate should balance the viral replication rate, we estimate both parameters from a large set of published experiments depleting CD8+T cells in SIV infected monkeys. This confirms that the killing rate can be much faster than is currently appreciated.

  • Visualization and sequencing of membrane remodeling leading to influenza virus fusion [PublishAheadOfPrint]

  • Protein-mediated membrane fusion is an essential step in many fundamental biological events including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling to the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane, led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved.

    IMPORTANCE Enveloped viruses employ specialized surface proteins to mediate fusion of cell and viral membranes resulting in the formation of pores through which the viral genetic material is transferred into the cell. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the 3-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

  • Targeted mutagenesis of guinea pig cytomegalovirus using CRISPR/Cas9-mediated gene editing [PublishAheadOfPrint]

  • The cytomegaloviruses are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kilobase pairs. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes, which necessitates the maintenance of the viral genome in E. coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes CRISPR/Cas9-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection was used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology driven repair. As CRISPR-Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate the genetic manipulation of low-passage or clinical CMV isolates.

    IMPORTANCE The cytomegalovirus genome is complex and viral adaptations to cell culture have complicated the study of infection in vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small animal model of congenital cytomegalovirus disease. CRISPR/Cas9-mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virally infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

  • Dynein Regulators Are Important For Ecotropic Murine Leukemia Virus Infection [PublishAheadOfPrint]

  • During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex, and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses such as murine leukemia virus (MLV), the identity of the cellular proteins involved in trafficking of the PIC in infection is unknown. To identify cellular proteins that interact with MLV PIC, we developed a replication-competent MLV virus in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNAi mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic but not amphotropic MLV reporter viruses. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by those retroviruses entering the cell via the ecotropic envelop pathway.

    IMPORTANCE Retroviruses must access the chromatin of the host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm -a crowded environment where diffusion is slow- is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We find that the preintegration complex of the murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of MLV preintegration complex.

  • Mosaic H5 hemagglutinin provides broad humoral and cellular immune responses against influenza viruses [PublishAheadOfPrint]

  • The most effective way to prevent influenza infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immune-depletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). When compared with a MVA construct expressing HA from influenza A/VN/1203/04, MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge; whereas, MVA-HA vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8+ T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine.

    IMPORTANCE Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000-500,000 deaths annually. Vaccines are still the main intervention to prevent infection but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

  • Measurements of intrahost viral diversity are extremely sensitive to systematic errors in variant calling [PublishAheadOfPrint]

  • With next generation sequencing technologies, it is now feasible to efficiently sequence patient-derived virus populations at a depth of coverage sufficient to detect rare variants. However, each sequencing platform has characteristic error profiles, and sample collection, target amplification, and library preparation are additional processes whereby errors are introduced and propagated. Many studies account for these errors by using ad hoc quality thresholds and/or previously published statistical algorithms. Despite common usage, the majority of these approaches have not been validated under conditions that characterize many studies of intrahost diversity. Here we use defined populations of influenza virus to mimic the diversity and titer typically found in patient-derived samples. We identified single nucleotide variants using two commonly used variant callers, DeepSNV and LoFreq. We found that the accuracy of these variant callers was lower than expected and exquisitely sensitive to input titer. Small reductions in specificity had a significant impact on the number of minority variants identified and subsequent measures of diversity. We were able to increase the specificity of DeepSNV to ggt;99.95% by applying an empirically validated set of quality thresholds. When applied to a set of influenza samples from a household-based cohort study, these changes resulted in a 10-fold reduction in measurements of viral diversity. We have made our sequence data and analysis code available so that others may improve on our work and use our dataset to benchmark their own bioinformatic pipelines. Our work demonstrates that inadequate quality control and validation can lead to significant overestimation of intrahost diversity.

    Importance Advances in sequencing technology have made it feasible to sequence patient-derived viral samples at a level sufficient for detection of rare mutations. These high-throughput, cost-effective methods are revolutionizing the study of within-host viral diversity. However, these techniques are error prone, and the methods commonly used to control for these errors have not been validated under the conditions that characterize patient-derived samples. Here we show that these conditions affect measurements of viral diversity. We found that the accuracy of previously benchmarked analysis pipelines were greatly reduced under patient-derived conditions. By carefully validating our sequencing analysis using known control samples, we were able to identify biases in our method and improve our accuracy to acceptable levels. Application of our modified pipeline to a set of influenza samples from a cohort study provide a realistic picture of intrahost diversity and suggest the need for rigorous quality control in such studies.

  • CD47 plays a role as a negative regulator in inducing protective immune responses to vaccination against influenza virus [PublishAheadOfPrint]

  • An integrin-associated protein CD47, which is a ligand for the inhibitory receptor signal regulatory protein aalpha;, is expressed on B and T cells as well as on most innate immune cells. However, the roles of CD47 in the immune responses to viral infection or vaccination remain unknown. We have investigated the role of CD47 in inducing humoral immune responses after intranasal infection with virus or immunization with influenza virus-like particles (VLP). Virus infection or vaccination with VLP containing hemagglutinin (HA) from A/PR8/34 influenza virus induced higher levels of antigen-specific IgG2c isotype dominant antibodies in CD47-deficient (CD47KO) mice than those in wild type (WT) mice. CD47KO mice with vaccination showed higher protective efficacy against lethal challenge, as evidenced by no loss in body weight and reduced lung viral titers compared to WT mice. In addition, inflammatory responses which include cytokine production, leukocyte infiltrates, and interferon gamma (IFN-)-producing CD4+ T cells as well as anti-inflammatory cytokine (IL-10) were reduced in the lung of vaccinated CD47KO mice after challenge with influenza virus. Analysis of lymphocytes indicated that GL7+ germinal center B cells were induced at higher levels in the draining lymph nodes of CD47KO mice as compared with those in WT mice. Notably, CD47KO mice exhibited significant increases in the numbers of antigen-specific memory B cells in spleens and plasma cells in bone marrow despite their lower levels of background IgG antibodies. Thus, these results suggest that CD47 plays a role as a negative regulator in inducing protective immune responses to influenza vaccination.

    IMPORTANCE Molecular mechanisms that control B cell activation to produce protective antibodies upon viral vaccination remain poorly understood. The CD47 molecule is known to be a ligand for the inhibitory receptor signal regulatory protein aalpha; and expressed on the surfaces of most immune cell types. CD47 was previously demonstrated to play an important role in modulating the migration of monocytes, neutrophils, polymorphonuclear neutrophils, and dendritic cells into the inflamed tissues. Results in this study demonstrate new roles of CD47 in negatively regulating the induction of protective IgG antibodies, germinal center B cells, and plasma cells secreting antigen-specific antibodies as well as macrophages upon influenza vaccination and challenge. As consequences, vaccinated CD47 deficient mice resulted in better control of influenza viral infection and enhanced protection. This study provides insights into understanding the regulatory functions of CD47 in inducing adaptive immunity to vaccination.

  • Small-molecule fusion inhibitors bind the pH-sensing SSP-GP2 subunit interface of the Lassa virus envelope glycoprotein [PublishAheadOfPrint]

  • Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photocrosslinking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever.

    IMPORTANCE Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition, and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

  • Both Cis- and Trans- Activities of the Foot-and-Mouth Disease Virus 3D Polymerase are Essential for Viral RNA Replication. [PublishAheadOfPrint]

  • The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a co-ordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e. via expression from a separate RNA molecule), whilst other are required in cis (i.e. expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA-polymerase (RdRp), 3D, are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically-active 3D molecules and those which build a replication complex. We report a novel non-enzymatic cis-acting function of 3D that is essential for viral genome replication. Using a FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans-acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localise to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. Together, this study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes and understanding this process can facilitate the development of novel therapeutic strategies. Many of the non-structural proteins involved in replication possess multiple functions in the viral life-cycle, some of which can be supplied to the replication complex from a separate genome (i.e. in trans) whilst other must originate from the template (i.e. in cis). Here, we present an analysis of cis- and trans-activities of the RNA-dependent RNA-polymerase, 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data furthers the understanding of genome replication of this important pathogen.

  • Transformation by Polyomavirus Middle T Antigen Involves A Unique Bimodal Interaction with the Hippo Effector YAP [PublishAheadOfPrint]

  • Murine polyomavirus has repeatedly provided insights into tumorigenesis, revealing such key control mechanisms as tyrosine phosphorylation and PI3K signaling. We recently demonstrated that polyomavirus ST antigen binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of MT antigen important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via shRNA reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type or YAP-binding mutant MT, promoted YAP phosphorylation at S127 and S381/397(YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with PP2A leading to YAP's dephosphorylation in the MT complex. It also leads to YAP's enrichment in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions.

    IMPORTANCE The highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study we show that MT signaling promotes YAP phosphorylation, loss from the nucleus and increased turn-over. Notably MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, the YAP bound to MT is dephosphorylated, stabilized and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.

  • Identification of diverse mycoviruses through metatranscriptomics characterization of the viromes of five major fungal plant pathogens [PublishAheadOfPrint]

  • Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the non-redundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts.

    IMPORTANCE Plant pathogenic fungi reduce crop yields, which affects food security worldwide. Plant-host resistance is considered a sustainable disease management option, but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence maybe welcome additions for mitigating plant diseases. By high throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungi-virus coevolution.

  • ISG12a restricts hepatitis C virus infection through ubiquitination-dependent degradation pathway [PublishAheadOfPrint]

  • Interferons (IFNs) restrict various kinds of viral infection via inducing hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high IFN-inducible gene ISG12a exhibits non-apoptotic antiviral effect to HCV infection. Viral NS5A protein is targeted specifically by ISG12a that mediates NS5A degradation via ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. SKP2 is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity.

    IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The study showed a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase SKP2 for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.

  • A novel open and infectious form of Echovirus 1 [PublishAheadOfPrint]

  • One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on earlier studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N-terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious Echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR Green II, it still contained most of its VP4, and it was able to bind to its receptor aalpha;2bbeta;1 integrin and showed high infectivity. A thermal assay further showed that the aalpha;2bbeta;1 integrin binding domain, I-domain, stabilized the virus particle. Finally, heating E1 particles to super-physiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate.

    IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It has been shown earlier that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to super-physiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure and it cannot be formed by heating. It still contains VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

  • Functional incompatibility between the generic NF-{kappa}B motif and a subtype-specific Sp1III element drives the formation of HIV-1 subtype C viral promoter [PublishAheadOfPrint]

  • Of the various genetic subtypes of HIV-1, HIV-2 and SIV, only in subtype C of HIV-1, a genetically variant NF-B binding site is found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-B and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be substituted by a corresponding TFBS variant.The variant NF-B motif binds NF-B with an affinity two-fold higher than that of the generic NF-B site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-B motif. An additional substitution of the Sp1III motif fully restores viral replication suggesting that the subtype C specific Sp1III has evolved to function with the variant, but not generic, NF-B motif. A change of only two base pairs in the central NF-B motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to TNF-aalpha; induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-B motif and a unique Sp1 site in the context of HIV-1 promoter. Our work provides important leads as per the evolution of HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency.

    Importance: Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes if any. Only in subtype C of HIV-1, two genetically distinct transcription factor binding sites are positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-B binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that subtype C viral promoter is not only stronger but also is endowed with a qualitative-gain-of-function advantage. The genetically variant NF-B, and the Sp1III motifs may be responsive to specific cell signal-pathways differentially, and these mechanisms must be examined.

  • CD8+ T cell breadth and ex vivo virus inhibition capacity distinguish between viremic controllers with and without protective HLA class I alleles [PublishAheadOfPrint]

  • The mechanisms of viral control and loss of viral control in chronically infected individuals with or without protective HLA class I alleles are not fully understood. We therefore characterized longitudinally the immunological and virological features that may explain divergence in disease outcome in 70 HIV-1 C-clade infected antiretroviral therapy (ART)-naïve South African adults, 35 of whom possessed protective HLA class I alleles. We demonstrate that, over five years of longitudinal study, 35% of individuals with protective HLA class I alleles lost viral control compared to none of individuals without protective HLA class I alleles (p=0.06). Sustained HIV-1 control in patients with protective HLA class I alleles was characteristically related to the breadth of HIV-1 CD8+ T-cell responses against Gag and enhanced ability of CD8+ T cells to suppress viral replication ex vivo. In some cases loss of virological control was associated with reduction in the total breadth of CD8+ T cell responses in the absence of differences in HIV-1-specific CD8+ T cell polyfunctionality or proliferation. In contrast, viremic controllers without protective HLA class I alleles possessed low breadth of HIV-1-specific CD8+ T cell responses characterized by reduced ability to suppress viral replication ex vivo. These data suggest that the control of HIV-1 in individuals with protective HLA class I alleles may be driven by broad CD8+ T cell responses with potent viral inhibitory capacity while control among individuals without protective HLA class I alleles may be more durable and mediated by CD8+ T cell independent mechanisms.

    Importance Host mechanisms of natural HIV-1 control are not fully understood. In a longitudinal study of antiretroviral therapy (ART)-naïve individuals, we show that those with protective HLA-I alleles subsequently experienced virologic failure compared to those without protective alleles. Among individuals with protective HLA-I alleles, viremic control was associated with broad CD8+ T cells that targeted the Gag protein, and CD8+ T cells from these individuals exhibited superior virus inhibition capacity. In individuals without protective HLA-I alleles, HIV-1-specific CD8+ T cell responses were narrow and poorly inhibited virus replication. These results suggest that broad, highly functional cytotoxic T cells (CTL) against the HIV-1 Gag protein are associated with control among those with protective HLA-I alleles and that loss of these responses eventually leads to viremia. A subset of individuals appears to have alternative, non-CTL mechanisms of viral control. These controllers may hold the key to an effective HIV vaccine.

  • Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells [PublishAheadOfPrint]

  • Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV back to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results using free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small pore (0.4 mmu;m) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large pore (3.0 mmu;m) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large pore membranes but not small pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4 dependent manner. Thus both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE.

    IMPORTANCE Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-cell contact rather than on particle release and re-entry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-cell contact rather than through cell-free particles. In addition, we seek to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: 1) myeloid cells generated from donated blood and 2) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e. monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium.

  • HIV frameshift site RNA stability correlates with frameshift efficiency and decreased viral infectivity [PublishAheadOfPrint]

  • HIV replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationship between the thermodynamic stability of the HIV-1 RNA frameshift site stem-loop, frameshift efficiency and infectivity using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-3 fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting there are no viral or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-Pol levels in cells and in virions support a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency, and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional, but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication.

    IMPORTANCE HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilize a panel of mutant HIV to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-3 fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest this effect might be exploited in the development of novel anti-viral strategies.

  • Uukuniemi virus as a tick-borne virus model [PublishAheadOfPrint]

  • In the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged in different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome the closest to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection is persistent. The glycoprotein GC was mainly highly-mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases PNGase F and Endo H. Treatment with bbeta;-mercaptoethanol did not impact the apparent molecular weight of GN. On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F and the electrophoresis mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per foci forming units differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells when investigating the cell biology of arbovirus transmission and entry into mammalian hosts.

    Importance Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research, not only on tick-borne phleboviruses, but on all viruses and other pathogens transmitted by ticks.

  • p53 is a Host Cell Regulator during Herpes Simplex Encephalitis [PublishAheadOfPrint]

  • p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knock-out reduced viral replication in the brains of mice and led to significantly reduced HSV-1 mortality due to encephalitis. These results suggest that p53 is an important host cell regulator for HSV-1 replication and pathogenesis in the central nervous system (CNS).

    IMPORTANCE HSV-1 causes sporadic encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered to be a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism that may suggest novel clinical treatment options for HSV-1 encephalitis.

  • Identification and Characterization of a Ribose 2-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales [PublishAheadOfPrint]

  • The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae and the recently recognized Mesoniviridae. RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2rrsquo; -O-MTase. Although bioinformatic analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated nidovirus (GAV), encodes the 2rrsquo; -O-MTase activity, although we could not detect 2rrsquo; -O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2rrsquo; -O-MTase, the roniviral 2rrsquo; -O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2rrsquo; -O-MTases and can only target the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with coronavirus, roniviral 2rrsquo; -O-MTase does not require a protein cofactor for stimulation of its activity, and differs in its preference for several biochemical parameters such as reaction temperature and pH. Furthermore, the ronivirus 2rrsquo; -O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family.

    IMPORTANCE: Methylation of the 5rrsquo; -cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as 2rrsquo; -O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales. In this study, we identified a 2rrsquo; -O-methyltransferase encoded by ronivirus and it shows common and unique features in comparison with that of coronaviruses. Ronivirus 2rrsquo; -O-MTase does not need a protein co-factor for MTase activity whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2rrsquo; -O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional link between roniviruses and coronaviruses.

  • Complete unique genome sequence, expression profile and salivary gland tissue tropism of the herpesvirus-7 homolog in pigtailed macaques [PublishAheadOfPrint]

  • Human herpesviruses HHV-6A, 6B and 7 are classified as roseoloviruses, and are highly prevalent in the human population. Roseolovirus reactivation in an immunocompromised host can cause severe pathologies. While the pathogenic potential of HHV-7 is unclear, it can reactivate HHV-6 from latency and thus contributes to severe pathological conditions associated with HHV-6. Because of the ubiquitous nature of roseoloviruses, their roles in such interactions and resulting pathological consequences have been difficult to study. Furthermore, the lack of a relevant animal model for HHV-7 infection has hindered a better understanding of its contribution to roseolovirus associated diseases.

    Using Next-Gen sequence analysis, we have characterized the unique genome of an uncultured novel pig-tailed macaque roseolovirus. Detailed genomic analysis revealed the presence of gene homologs to all 84 known HHV-7 ORFs. Phylogenetic analysis confirmed that the virus is a macaque homolog of HHV-7, which we have provisionally named MneHV7.

    Using high-throughput RNA sequencing, we observed that the salivary gland tissue samples from nine different macaques had distinct MneHV7 gene expression patterns and that the overall number of viral transcripts correlated with viral loads in parotide tissue and saliva. Immunohistochemistry staining confirmed that like HHV-7, MneHV7 exhibits natural tropism for salivary gland ductal cells. We also observed staining for MneHV7 in peripheral nerve ganglia present in salivary gland tissues suggesting that HHV-7 may also have a tropism for the peripheral nervous system.

    Our data demonstrate that MneHV7-infected macaques represent a relevant animal model that may help clarify the causality between roseolovirus reactivation and diseases.

    Importance Human herpesviruses HHV-6A, 6B and 7 are classified as Roseoloviruses. We have recently discovered that pigtailed macaques are naturally infected with viral homologs of HHV-6 and HHV-7 which we provisionally named MneHV6 and MneHV7, respectively. In this study we confirm that MneHV7 is genetically and biologically similar to its human counterpart HHV-7. We determine the complete unique MneHV7 genome sequence and provide a comprehensive annotation of all genes. We also characterize viral transcription profiles in salivary glands from naturally infected macaques. We show that broad transcriptional activity across most of the viral genome is associated with high viral loads in infected parotids and that late viral protein expression is detected in salivary duct cells and peripheral nerve ganglia. Our study provides new insights into the natural behavior of an extremely prevalent virus and establishes a basis for subsequent investigations of the mechanisms that cause HHV-7 reactivation and associated disease.

  • Requirement of HSP90 chaperoning in addition to phosphoprotein for folding but not for supporting enzymatic activities of measles and Nipah virus L polymerases [PublishAheadOfPrint]

  • Non-segmented negative-stranded RNA viruses, or Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have co-evolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes L protein, through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-DMAG as specific inhibitors of the cellular heat shock protein 90 (HSP90), we report that the efficient chaperoning of L by HSP90 requires P in the case of measles, Nipah and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N-terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L as evidenced by a luciferase reporting insert fused within measles virus L. HSP90 acts as a true chaperon: its activity is transient and dispensable for the activity of measles and Nipah polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order.

    IMPORTANCE Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus the successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, whilst P protein binds to the N-terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved for several members of the Mononegavirales order including Nipah and vesicular stomatitis virus.

  • Arteriviruses, pegiviruses, and lentiviruses are common among wild African monkeys [PublishAheadOfPrint]

  • Non-human primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV, family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks, Chlorocebus cynosuros) and South Africa (vervet monkeys, Chlorocebus pygerythrus). We examine several aspects of infection including viral load, genetic diversity, evolution, geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys.

    IMPORTANCE Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by HIV in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys co-infected with pegiviruses, possibly because SIV causes little-to-no disease in these hosts.

  • HIV-1 Vpu Antagonizes CD317/tetherin by Adaptor protein-1-Mediated Exclusion from Virus Assembly Sites [PublishAheadOfPrint]

  • The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. On the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a di-serine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1 and CD317/tetherin in this process.

    Importance CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

  • Initiation of antiretroviral therapy restores CD4+ TSCM homeostasis in SIV-infected macaques [PublishAheadOfPrint]

  • Treatment of human immunodeficiency virus (HIV) infection with antiretroviral therapy (ART) has significantly improved prognosis. Unfortunately, interruption of ART almost invariably results in viral rebound, attributed to a pool of long-lived, latently-infected cells. Based on their longevity and proliferative potential, CD4+ Tmemory stem cells (TSCM) have been proposed as an important site of HIV persistence. In a previous study, we found that, in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM), CD4+ TSCM are preserved in number but show (i) a decrease in the frequency of CCR5+ cells, (ii) an expansion of the fraction of proliferating Ki-67+ cells, and (iii) high levels of SIV-DNA. To understand the impact of ART on both CD4+ TSCM homeostasis and virus persistence, we conducted a longitudinal analysis of these cells in the blood and lymph nodes of twenty-five SIV-infected RM. We found that ART induced a significant restoration of CD4+CCR5+ TSCM in both blood and lymph nodes, and a reduction in the fraction of proliferating CD4+Ki-67+ TSCM in blood (but not lymph nodes). Importantly, we found that the level of SIV-DNA in CD4+ transitional memory (TTM-) and effector memory (TEM-) T cells declined ~100-fold after ART in both blood and lymph nodes, while the level of SIV-DNA in CD4+ TSCM and central memory T cells (TCM-) did not significantly change. These data suggest that ART is effective at partially restoring CD4+ TSCM homeostasis and the observed stable level of virus in TSCM supports the hypothesis that these cells are a critical contributor to SIV persistence.

    IMPORTANCE: Understanding the role of various CD4+ T cell memory subsets in immune homeostasis and HIV/SIV persistence during antiretroviral therapy (ART) is critical to effectively treat and cure HIV infection. T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into other memory T cell subsets, such as central and transitional memory T cells (TCM and TTM, respectively). CD4+ TSCM are disrupted, but not depleted during pathogenic SIV infection. We find ART is partially effective at restoring CD4+ TSCM homeostasis and that SIV-DNA harbored within this subset contracts more slowly compared to virus harbored in shorter-lived subsets, such as TTM and effector memory (TEM). Because of their ability to persist long-term in an individual, understanding the dynamics of virally infected CD4+ TSCM during suppressive ART is important for future therapeutic interventions aimed at modulating immune activation and purging the HIV reservoir.

  • Identification of a functionally relevant AAV Rep68 oligomeric interface [PublishAheadOfPrint]

  • The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins Rep78 and Rep68 are remarkably multi-functional and display a range of biochemical activities, including DNA binding, nicking and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication and genomic integration. Structurally, the Rep proteins share a AAA+ domain characteristic of the SF3 family of helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) domain that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report we describe an oligomeric interface formed by Rep68 and demonstrate how disrupting this interface has drastic effects both on the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helical bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle.

    Importance The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood, however Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

  • Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes [PublishAheadOfPrint]

  • The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete and recycle a chemically diverse set of bile acids with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC) glycochenodeoxycholic acid and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anti-cytomegalovirus activity required active import of bile acids via sodium-taurocholate cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which IFN-aalpha; lacks antiviral activity, physiological TCDC concentrations were similarly effective as interferon (IFN)-. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells.

    Importance Cytomegaloviruses are members of bbeta;-herpesvirinae. Primary infection leads to latency from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

  • Structural and biochemical analyses of swine MHC class I complexes and prediction of the epitope map in important influenza A strains [PublishAheadOfPrint]

  • The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing of 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. The SLA-3*hs0202 binding with sbbeta;2m and a KMNTQFTAV (HA-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune response. Notably, we found that different HA-KMN9 conformations are caused not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG) but also by the skewing of aalpha;1 and aalpha;2 helixes forming the PBG. In addition, alanine scanning and CD spectra confirmed that the B, D and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC I) structures, the fundamental motif for SLA-3*hs0202 was determined as llsquo;X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I)rrsquo; by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

    IMPORTANCE We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previously findings showing that variable peptide conformations are only caused by the flexibility of the side chains in the groove, the skewing of the aalpha;1 and aalpha;2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 as llsquo;X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I)rrsquo; based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

  • Identification and validation of small molecules that enhance recombinant Adeno-associated virus transduction following high throughput screen [PublishAheadOfPrint]

  • While the recent success of AAV-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2396 compounds. We identified 13 compounds that were capable of enhancing transduction, 12 of which demonstrated vector-specific effects and one that could also enhance vector-independent transgene expression. Many of these compounds shared similar properties and could be categorized into five groups: Epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors. In vivo analysis of rAAV2 and all of the clinically-relevant compounds revealed that, consistent with our in vitro results, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectors in vivo using an AAV8 capsid, which is known to exhibit robust liver tropism. Consistent with our in vitro results, teniposide co-administration greatly enhanced fragmented rAAV8 transduction at 48h and 8 days. This study provides a foundation based on rAAV small molecule screen methodology which is ideally studied for more diverse libraries of compounds that can be tested for potentiating rAAV transduction.

    IMPORTANCE This study seeks to enhance the ability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs was tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50-100 times better using teniposide, which is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy which could result in a safer and effective treatments for diverse acquired or genetic diseases.

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