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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]

  • Recognizing the Top 25 Peer Reviewers for the Journal of Virology [Editorials]

  • Acknowledgment of Ad Hoc Reviewers [Editorials]

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

  • Rationally Designed Vaccines Targeting the V2 Region of HIV-1 gp120 Induce a Focused, Cross-Clade-Reactive, Biologically Functional Antibody Response [Vaccines and Antiviral Agents]

  • Strong antibody (Ab) responses against V1V2 epitopes of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, simian-human immunodeficiency virus (SHIV), and simian immunodeficiency virus (SIV). In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (i) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (ii) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (iii) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (iv) remained detectable gge;1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally designed epitope scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting, rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV, and SHIV.

    IMPORTANCE Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV, and SHIV.

  • Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits [Vaccines and Antiviral Agents]

  • The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the bbeta;-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope.

    IMPORTANCE The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

  • Altering the Anti-inflammatory Lipoxin Microenvironment: a New Insight into Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis [Cellular Response to Infection]

  • Lipoxins are host anti-inflammatory molecules that play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and its associated diseases has been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. This study documents how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. The presence of the lipoxin A4 receptor/formyl peptidyl receptor (ALX/FPR) in KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing the levels of NF-B, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene deletion revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral microRNA (miRNA) cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. This study provides a new insight into the potential treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin.

    IMPORTANCE KSHV infection has been shown to upregulate several host proinflammatory factors, which aid in its survival and pathogenesis. The influence of KSHV infection on anti-inflammatory molecules is not well studied. Since current treatment methods for KS and PEL are fraught with unwanted side effects and low efficiency, the search for new therapeutics is therefore imperative. The use of nature's own molecule lipoxin as a drug is promising. This study opens up new domains in KSHV research focusing on how the virus modulates lipoxin secretion and warrants further investigation of the therapeutic potential of lipoxin using in vitro cell models for KS and PEL.

  • Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection [Virus-Cell Interactions]

  • The human rhinovirus (HRV) 3C and 2A proteases (3Cpro and 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cpro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Apro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cpro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cpro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Apro fusion proteins, we demonstrate formally that 2Apro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cpro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Apro activity and not 3Cpro activity, which is observed only later in infection. The results thus define the temporal activities of 2Apro and 3CD/3Cpro activities in HRV serotype16 infection.

    IMPORTANCE The human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

  • Bipartite Network Analysis of the Archaeal Virosphere: Evolutionary Connections between Viruses and Capsidless Mobile Elements [Genetic Diversity and Evolution]

  • Archaea and particularly hyperthermophilic crenarchaea are hosts to many unusual viruses with diverse virion shapes and distinct gene compositions. As is typical of viruses in general, there are no universal genes in the archaeal virosphere. Therefore, to obtain a comprehensive picture of the evolutionary relationships between viruses, network analysis methods are more productive than traditional phylogenetic approaches. Here we present a comprehensive comparative analysis of genomes and proteomes from all currently known taxonomically classified and unclassified, cultivated and uncultivated archaeal viruses. We constructed a bipartite network of archaeal viruses that includes two classes of nodes, the genomes and gene families that connect them. Dissection of this network using formal community detection methods reveals strong modularity, with 10 distinct modules and 3 putative supermodules. However, compared to similar previously analyzed networks of eukaryotic and bacterial viruses, the archaeal virus network is sparsely connected. With the exception of the tailed viruses related to bacteriophages of the order Caudovirales and the families Turriviridae and Sphaerolipoviridae that are linked to a distinct supermodule of eukaryotic and bacterial viruses, there are few connector genes shared by different archaeal virus modules. In contrast, most of these modules include, in addition to viruses, capsidless mobile elements, emphasizing tight evolutionary connections between the two types of entities in archaea. The relative contributions of distinct evolutionary origins, in particular from nonviral elements, and insufficient sampling to the sparsity of the archaeal virus network remain to be determined by further exploration of the archaeal virosphere.

    IMPORTANCE Viruses infecting archaea are among the most mysterious denizens of the virosphere. Many of these viruses display no genetic or even morphological relationship to viruses of bacteria and eukaryotes, raising questions regarding their origins and position in the global virosphere. Analysis of 5,740 protein sequences from 116 genomes allowed dissection of the archaeal virus network and showed that most groups of archaeal viruses are evolutionarily connected to capsidless mobile genetic elements, including various plasmids and transposons. This finding could reflect actual independent origins of the distinct groups of archaeal viruses from different nonviral elements, providing important insights into the emergence and evolution of the archaeal virome.

  • Herpes Simplex Virus 1 Tegument Protein UL41 Counteracts IFIT3 Antiviral Innate Immunity [Virus-Cell Interactions]

  • The interferon-induced protein with tetratricopeptide repeat 3 (IFIT3 or ISG60) is a host-intrinsic antiviral factor that restricts many instances of DNA and RNA virus replication. Herpes simplex virus 1 (HSV-1), a DNA virus bearing a large genome, can encode many viral proteins to counteract the host immune responses. However, whether IFIT3 plays a role upon HSV-1 infection is little known. In this study, we show for the first time that HSV-1 tegument protein UL41, a viral endoribonuclease, plays an important role in inhibiting the antiviral activity of IFIT3. Here, we demonstrated that ectopically expressed IFIT3 could restrict the replication of vesicular stomatitis virus (VSV) but had little effect on the replication of wild-type (WT) HSV-1. Further study showed that WT HSV-1 infection downregulated the expression of IFIT3, and ectopic expression of UL41, but not the immediate-early protein ICP0, notably reduced the expression of IFIT3. The underlying molecular mechanism was that UL41 diminished the accumulation of IFIT3 mRNA to abrogate its antiviral activity. In addition, our results illustrated that ectopic expression of IFIT3 inhibited the replication of UL41-null mutant virus (R2621), and stable knockdown of IFIT3 facilitated its replication. Taking these findings together, HSV-1 was shown for the first time to evade the antiviral function of IFIT3 via UL41.

    IMPORTANCE The tegument protein UL41 of HSV-1 is an endoribonuclease with the substrate specificity of RNase A, which plays an important role in viral infection. Upon HSV-1 infection, interferons are critical cytokines that regulate immune responses against viral infection. Host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3; however, whether IFIT3 plays a role during HSV-1 infection is still unknown. Our data show for the first time that IFIT3 has little effect on HSV-1 replication, as UL41 decreases the accumulation of IFIT3 mRNA and subverts its antiviral activity. This study identifies IFIT3 as a novel target of the tegument protein UL41 and provides new insight into HSV-1-mediated immune evasion.

  • Antagonism of BST-2/Tetherin Is a Conserved Function of the Env Glycoprotein of Primary HIV-2 Isolates [Virus-Cell Interactions]

  • Although HIV-2 does not encode a vpu gene, the ability to antagonize bone marrow stromal antigen 2 (BST-2) is conserved in some HIV-2 isolates, where it is controlled by the Env glycoprotein. We previously reported that a single-amino-acid difference between the laboratory-adapted ROD10 and ROD14 Envs controlled the enhancement of virus release (referred to here as Vpu-like) activity. Here, we investigated how conserved the Vpu-like activity is in primary HIV-2 isolates. We found that half of the 34 tested primary HIV-2 Env isolates obtained from 7 different patients enhanced virus release. Interestingly, most HIV-2 patients harbored a mixed population of viruses containing or lacking Vpu-like activity. Vpu-like activity and Envelope functionality varied significantly among Env isolates; however, there was no direct correlation between these two functions, suggesting they evolved independently. In comparing the Env sequences from one HIV-2 patient, we found that similar to the ROD10/ROD14 Envs, a single-amino-acid change (T568I) in the ectodomain of the TM subunit was sufficient to confer Vpu-like activity to an inactive Env variant. Surprisingly, however, absence of Vpu-like activity was not correlated with absence of BST-2 interaction. Taken together, our data suggest that maintaining the ability to antagonize BST-2 is of functional relevance not only to HIV-1 but also to HIV-2 as well. Our data show that as with Vpu, binding of HIV-2 Env to BST-2 is important but not sufficient for antagonism. Finally, as observed previously, the Vpu-like activity in HIV-2 Env can be controlled by single-residue changes in the TM subunit.

    IMPORTANCE Lentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode a vpu gene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2 in some isolates. Here, we cloned multiple Env sequences from 7 HIV-2-infected patients and found that about half were able to antagonize BST-2. Importantly, most HIV-2 patients harbored a mixed population of viruses containing or lacking the ability to antagonize BST-2. In fact, in comparing Env sequences from one patient combined with site-directed mutagenesis, we were able to restore BST-2 antagonism to an inactive Env protein by a single-amino-acid change. Our data suggest that targeting BST-2 by HIV-2 Env is a dynamic process that can be regulated by simple changes in the Env sequence.

  • The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies [Virus-Cell Interactions]

  • The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism.

    IMPORTANCE Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction.

  • TRIM5{alpha} Resistance Escape Mutations in the Capsid Are Transferable between Simian Immunodeficiency Virus Strains [Pathogenesis and Immunity]

  • TRIM5aalpha; polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5aalpha;-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5aalpha; restriction when it was passaged in rhesus macaques expressing restrictive TRIM5aalpha; alleles. Here we generated TRIM5aalpha;-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5aalpha; alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5aalpha; genotypes as efficiently as in macaques with permissive TRIM5aalpha; genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5aalpha; restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies.

    IMPORTANCE Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5aalpha; alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5aalpha; genotype and thus will eliminate the confounding effects of TRIM5aalpha; in vaccine studies.

  • Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus [Virus-Cell Interactions]

  • Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors.

    IMPORTANCE Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

  • Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression [Virus-Cell Interactions]

  • The role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein.

    IMPORTANCE This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

  • Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection [Cellular Response to Infection]

  • Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo.

    IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive "recall" or "anamnestic" response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

  • HIV-1 Glycan Density Drives the Persistence of the Mannose Patch within an Infected Individual [Vaccines and Antiviral Agents]

  • The HIV envelope glycoprotein (Env) is extensively modified with host-derived N-linked glycans. The high density of glycosylation on the viral spike limits enzymatic processing, resulting in numerous underprocessed oligomannose-type glycans. This extensive glycosylation not only shields conserved regions of the protein from the immune system but also acts as a target for anti-HIV broadly neutralizing antibodies (bnAbs). In response to the host immune system, the HIV glycan shield is constantly evolving through mutations affecting both the positions and numbers of potential N-linked glycosylation sites (PNGSs). Here, using longitudinal Env sequences from a clade C-infected individual (CAP256), we measured the impact of the shifting glycan shield during HIV infection on the abundance of oligomannose-type glycans. By analyzing the intrinsic mannose patch from a panel of recombinant CAP256 gp120s displaying high protein sequence variability and changes in PNGS number and positioning, we show that the intrinsic mannose patch persists throughout the course of HIV infection and correlates with the number of PNGSs. This effect of the glycan density on the processing state was also supported by the analysis of a cross-clade panel of recombinant gp120 glycoproteins. Together, these observations underscore the importance of glycan clustering for the generation of carbohydrate epitopes for anti-HIV bnAbs. The persistence of the intrinsic mannose patch over the course of HIV infection further highlights this epitope as an important target for HIV vaccine strategies.

    IMPORTANCE Development of an HIV vaccine is critical for control of the HIV pandemic, and elicitation of broadly neutralizing antibodies (bnAbs) is likely to be a key component of a successful vaccine response. The HIV envelope glycoprotein (Env) is covered in an array of host-derived N-linked glycans often referred to as the glycan shield. This glycan shield is a target for many of the recently isolated anti-HIV bnAbs and is therefore under constant pressure from the host immune system, leading to changes in both glycan site frequency and location. This study aimed to determine whether these genetic changes impacted the eventual processing of glycans on the HIV Env and the susceptibility of the virus to neutralization. We show that despite this variation in glycan site positioning and frequency over the course of HIV infection, the mannose patch is a conserved feature throughout, making it a stable target for HIV vaccine design.

  • Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3 [Cellular Response to Infection]

  • A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-aalpha; and IFN-bbeta;. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3.

    IMPORTANCE The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.

  • Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses [Pathogenesis and Immunity]

  • A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.

    IMPORTANCE Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

  • Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo [Pathogenesis and Immunity]

  • Human immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-specific CD8+ T cells are typically largely excluded from lymphoid B cell follicles, where HIV- and SIV-producing cells are most highly concentrated, indicating that B cell follicles are somewhat of an immunoprivileged site. To gain insights into virus-specific follicular CD8+ T cells, we determined the location and phenotype of follicular SIV-specific CD8+ T cells in situ, the local relationship of these cells to Foxp3+ cells, and the effects of CD8 depletion on levels of follicular SIV-producing cells in chronically SIV-infected rhesus macaques. We found that follicular SIV-specific CD8+ T cells were able to migrate throughout follicular areas, including germinal centers. Many expressed PD-1, indicating that they may have been exhausted. A small subset was in direct contact with and likely inhibited by Foxp3+ cells, and a few were themselves Foxp3+. In addition, subsets of follicular SIV-specific CD8+ T cells expressed low to medium levels of perforin, and subsets were activated and proliferating. Importantly, after CD8 depletion, the number of SIV-producing cells increased in B cell follicles and extrafollicular areas, suggesting that follicular and extrafollicular CD8+ T cells have a suppressive effect on SIV replication. Taken together, these results suggest that during chronic SIV infection, despite high levels of exhaustion and likely inhibition by Foxp3+ cells, a subset of follicular SIV-specific CD8+ T cells are functional and suppress viral replication in vivo. These findings support HIV cure strategies that augment functional follicular virus-specific CD8+ T cells to enhance viral control.

    IMPORTANCE HIV- and SIV-specific CD8+ T cells are typically largely excluded from lymphoid B cell follicles, where virus-producing cells are most highly concentrated, suggesting that B cell follicles are somewhat of an immunoprivileged site where virus-specific CD8+ T cells are not able to clear all follicular HIV- and SIV-producing cells. To gain insights into follicular CD8+ T cell function, we characterized follicular virus-specific CD8+ T cells in situ by using an SIV-infected rhesus macaque model of HIV. We found that subsets of follicular SIV-specific CD8+ T cells are able to migrate throughout the follicle, are likely inhibited by Foxp3+ cells, and are likely exhausted but that, nonetheless, subsets are likely functional, as they express markers consistent with effector function and show signs of suppressing viral replication in vivo. These findings support HIV cure strategies that increase the frequency of functional follicular virus-specific CD8+ T cells.

  • The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) particles are described as lipoviroparticles which are released similarly to very-low-density lipoproteins (VLDLs). However, the release mechanism is still poorly understood; the canonical endoplasmic reticulum-Golgi intermediate compartment (ERGIC) pathway as well as endosome-dependent release has been proposed. Recently, the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo-synthesized lipoviroparticles is released by the infected cell. To investigate the relevance of multivesicular bodies (MVBs) for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover, an mCherry-tagged HCV variant was used. Conditions were established that enable U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions, significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density, reflecting a misloading with lipids. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation.

    IMPORTANCE There are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles that are released, raising the question about the fate of the nonreleased particles. Moreover, the relevance of the endosomal pathway for the release of HCV is under debate. Use of the MVB (multivesicular body) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for viral morphogenesis and release. It was revealed that infectious, fully assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway. Our study describes a so-far-unprecedented cross talk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of nonreleased particles.

  • Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12 [Virus-Cell Interactions]

  • Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60PSPMA65), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25DLLTEDPPPY34, which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70RREPP74) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70AAAAA74) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p1270nndash;74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61SPIASRLRGRR71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25DLLTEDPPPY34 motif in the N terminus suppresses the ability to tether.

    IMPORTANCE This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p1261nndash;71. Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.

  • Phosphorylation Requirement of Murine Leukemia Virus p12 [Virus-Cell Interactions]

  • The p12 protein of murine leukemia virus (MLV) Gag is associated with the preintegration complex (PIC), and mutants of p12 (PM14) exhibit defects in nuclear entry/retention. Mutants of the phosphorylated serine 61 also have been reported to have defects in the early life cycle. Here we show that a phosphorylated peptide motif derived from human papillomavirus 8 (HPV-8), the E2 hinge region including residues 240 to 255, can functionally replace the main phosphorylated motif of MLV p12 and can rescue the viral titer of a strain with the lethal p12-PM14 mutation. Complementation with the HPV-8 E2 hinge motif generated multiple second-site mutations in live viral passage assays. Additional p12 phosphorylation sites were detected, including the late domain of p12 (PPPY) as well as the late domain/protease cleavage site of matrix (LYPAL), by mass spectrometry and Western blotting. Chromatin binding of p12-green fluorescent protein (GFP) fusion protein and functional complementation of p12-PM14 occurred in a manner independent of the E2 hinge region phosphorylation. Replacement of serine 61 by alanine within the minimal tethering domain (61SPMASRLRGRR71) maintained tethering, but in the context of the full-length p12, mutants with substitutions in S61 remained untethered and lost infectivity, indicating phosphorylation of p12 serine 61 functions to temporally regulate early and late p12 functions.

    IMPORTANCE The p12 protein, required for both early and late viral functions, is the predominant phosphorylated viral protein of Moloney MLV and is required for virus viability. Our studies indicate that the N terminus of p12 represses the early function of the chromatin binding domain and that deletion of the N terminus activates chromatin binding in the wild-type Moloney MLV p12 protein. Mass spectrometry and mutagenesis studies suggest that phosphorylation of both the repression domain and the chromatin binding domain acts to temporally regulate this process at the appropriate stages during infection.

  • Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1 [Structure and Assembly]

  • Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ~450-AAring;-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.

    IMPORTANCE Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

  • TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus [Virus-Cell Interactions]

  • The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV.

    IMPORTANCE Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

  • Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification [Genetic Diversity and Evolution]

  • Knowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation.

    IMPORTANCE Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.

  • Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India [Cellular Response to Infection]

  • Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DRnndash; CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN- unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN- by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN- unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue.

    IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN- in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

  • Correction for Abrisch et al., Infection by Herpes Simplex Virus 1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome [Author Corrections]

  • Correction for Eduardo-Correia et al., ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection [Author Corrections]

  • Correction for Royo et al., Differential Induction of Apoptosis, Interferon Signaling, and Phagocytosis in Macrophages Infected with a Panel of Attenuated and Nonattenuated Poxviruses [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Inhibition of an aquatic rhabdovirus demonstrates promise of a broad-spectrum antiviral for use in aquaculture [PublishAheadOfPrint]

  • Many enveloped viruses cause devastating disease in aquaculture, resulting in significant economic impact. LJ001 is a broad-spectrum antiviral compound that inhibits enveloped viral infections by specifically targeting phospholipids in the lipid bilayer via production of singlet oxygen (1O2). This stabilizes positive curvature and decreases membrane fluidity, which inhibits viral-cell membrane fusion during viral entry. Based on previous mammalian studies and the requirement of light for activation of LJ001, we hypothesized that LJ001 may be useful as a preventative and/or therapeutic agent for enveloped viral infections in aquaculture. Here, we report that LJ001 was more stable with a prolonged inhibitory half-life at relevant aquaculture temperatures (15ddeg;C), as compared to mammalian studies at 37ddeg;C. When LJ001 was pre-incubated with our model virus, infectious hematopoietic necrosis virus (IHNV), infectivity was significantly inhibited in vitro (using the EPC fish cell line) and in vivo (using rainbow trout fry) in a dose-dependent and time-dependent manner. While horizontal transmission of IHNV in a static co-habitation challenge model was reduced by LJ001, transmission was not completely blocked at established antiviral doses. Therefore, LJ001 may be best suited as a therapeutic for aquaculture settings that include viral infections with lower viral shedding rates than IHNV, or where higher viral titers are required to initiate infection of naïve fish. Importantly, our data also suggests that LJ001-inactivated IHNV elicited an innate immune response in the rainbow trout host, making LJ001 potentially useful for future vaccination approaches.

    IMPORTANCE Viral diseases in aquaculture are challenging because there are few preventative measures and/or treatments. Broad-spectrum antivirals are highly sought after and studied because they target common components of viruses. In our studies, we used LJ001, a broad-spectrum antiviral compound that specifically inhibits enveloped viruses. We used the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) as a model to study aquatic enveloped virus diseases and their inhibition. We demonstrated inhibition of IHNV by LJ001 both in cell culture as well as in live fish. Additionally, we showed that LJ001 inhibited transmission of IHNV from infected fish to healthy fish, which lays the groundwork for using LJ001 as a possible therapeutic for aquatic viruses. Our results also suggest that virus inactivated by LJ001 induces an immune response, showing potential for future preventative (e.g. vaccine) applications.

  • A protective role for IL-1 signaling during mouse adenovirus type 1-induced encephalitis [PublishAheadOfPrint]

  • IL-1bbeta;, an inflammatory cytokine and IL-1 receptor ligand, has diverse activities in the brain. We examined whether IL-1 signaling contributes to the encephalitis observed in mouse adenovirus type 1 (MAV-1) infection, using mice lacking the IL-1 receptor, Il1r1-/-. Il1r1-/- mice had lower survival, higher disruption of the blood-brain barrier (BBB), higher brain viral loads, and higher brain inflammatory cytokines and chemokines than control C57BL/6 mice. We also examined infections of mice defective in IL-1bbeta; production (Pycard-/-mice) and mice defective in trafficking of TLRs to the endosome (Unc93b1-/- mice). Pycard-/- and Unc93b1-/- mice had lower survival (similar to Il1r1-/- mice) than control mice, but unlike Il1r1-/- mice, did not have increased brain viral loads or BBB disruption. Based on brain cytokine levels, MAV-1-infected Unc93b1-/- mice had a very different inflammatory profile from infected Il1r1-/- and Pycard-/- mice. Histological examination demonstrated pathological findings consistent with encephalitis in control and knockout mice; however, intranuclear viral inclusions were seen only in Il1r1-/- mice. A time course of infection of control and Il1r1-/- mice evaluating the kinetics of viral replication and cytokine production revealed differences between the mouse strains primarily at 7 - 8 days after infection, when mice began succumbing to MAV-1 infection. In the absence of IL-1 signaling, we noted an increase in the transcription of type I interferon (IFN)-stimulated genes. Together these results indicate that IL-1 signaling is important during MAV-1 infection and suggest that in its absence, increased IFN-bbeta; signaling may result in increased neuroinflammation.

    IMPORTANCE The investigation of encephalitis pathogenesis produced by different viruses is needed to characterize virus and host-specific factors that contribute to disease. MAV-1 produces viral encephalitis in its natural host, providing a good model for studying factors involved in encephalitis development. We investigated the role of IL-1 signaling during MAV-1-induced encephalitis. Unexpectedly, the lack of IL-1 signaling increased the mortality and inflammation in mice infected with MAV-1. Also there was an increase in the transcription of type I IFN-stimulated genes that correlated with the observed increased mortality and inflammation. The findings highlight the complex nature of encephalitis and suggests that IL-1 has a protective effect for the development of MAV-1-induced encephalitis.

  • Accumulation of Pol mutations selected by HLA-B*52:01-C*12:02 protective haplotype-restricted CTLs causes low plasma viral load due to low viral fitness of mutant viruses [PublishAheadOfPrint]

  • HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel CTL epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and, thus, reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated.

    IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals.

  • The human cytomegalovirus IE1 protein antagonizes PML nuclear body mediated intrinsic immunity via the inhibition of PML de novo SUMOylation [PublishAheadOfPrint]

  • PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV), however, this intrinsic defense is counteracted by the immediate-early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML, however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160 thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil mediated PML dimerization we propose that IE1 either affects PML autoSUMOylation by directly abrogating PML E3 ligase function or by preventing the access to SUMO sites. Thus, our data suggest a novel mechanism how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation.

    IMPORTANCE The human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by a subnuclear structure termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism how IE1 antagonizes PML-NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility to develop small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.

  • CXCR6-mediated SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections [PublishAheadOfPrint]

  • African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of SIV, which do not progress to AIDS when infected with their species-specific viruses. SIV natural hosts express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo, and blocking CCR5 does not prevent ex vivo infection of PBMC from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro. Here we investigated use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and ten candidate coreceptors. Species-matched CXCR6, CCR5 and GPR15 mediated robust entry into transfected cells by pseudotypes carrying the SIVagmSab92018ivTF Env, with lower level entry through GPR1 and APJ. We cloned genetically divergent envs from plasma of two wild-infected sabaeus and found similar coreceptor use patterns. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had greater impact than the CCR5 blocker maraviroc, confirming CXCR6 use in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus/host coevolution is use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4+ T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function.

    Importance Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic HIV-1/human and SIVmac/macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4+ cells expressing the SIV coreceptor CCR5. In this study, we demonstrate efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm that infects sooty mangabeys and SIVagmVer that infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which CCR5 expression is low, use of CXCR6 or other coreceptors to mediate infection may target SIV towards distinct cell populations that are able to support high viral replication without causing loss of CD4+ T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infection.

  • Genetic Variability of Myxoma Virus Genomes [PublishAheadOfPrint]

  • Myxomatosis is a recurrent problem on rabbit farms throughout Europe despite the success of vaccines. To identify gene variations of field and vaccine strains that may be responsible for changes in virulence, immunomodulation, and immunoprotection, the genomes of 6 Myxoma virus strains (MYXV) were sequenced: German field isolates Munich-1, FLI-H, 2604, 3207, vaccine strain MAV, and challenge strain ZA. The analyzed genomes ranged from 147.6 kb (strain MAV) to 161.8 kb (strain 3207) in size. All sequences were affected by several mutations, covering 24-93 open reading frames (ORFs) and resulted in amino acid (aa) substitutions, insertions, or deletions. Only strains Munich-1 and MAV revealed the deletion of 10 (M007L-M015L) and 11 (M007L-M008.1L, M149R-M008.1R) ORFs, respectively. Major differences were observed in the 27 immunomodulatory proteins encoded by MYXV. Compared to the reference strain Lausanne, strains FLI-H, 2604, 3207, and ZA showed the highest aa identity (ggt;98.4%). In strains Munich-1 and MAV the deletion of 5 and 10 ORFs, respectively, was observed, encoding immunomodulatory proteins with ankyrin repeats or members of the family of serine protease inhibitors. Furthermore, putative immunodominant surface proteins with homology to vaccinia virus (VACV) were investigated in the sequenced strains. Only strain MAV revealed aa substitutions and frameshift mutations above average. Finally, we performed recombination analysis and found signs of recombination in vaccine strain MAV. Phylogenetic analysis showed a close relationship of strain MAV and the MSW strain of Californian MYXV. However, in a challenge model strain MAV provided full protection against lethal challenges with strain ZA.

    Importance Myxoma virus (MYXV) is pathogenic for European rabbits and two North American species. Due to sophisticated strategies in immune evasion and oncolysis, MYXV is an important model virus for immunological and pathological research. In its natural hosts MYXV causes a benign infection, whereas in European rabbits it causes the lethal disease myxomatosis. Since introduction of MYXV into Australia and Europe for biological control of European rabbits in the 1950s, a co-evolution of host and pathogen has started, selecting for attenuated virus strains and increased resistance in rabbits. Evolution of viruses is a continuous process and influences the protective potential of vaccines. In our analyses, we sequenced 6 MYXV field, challenge, and vaccine strains. We focused on genes encoding proteins involved in virulence, host-range, immunomodulation, and envelope composition. Genes affected most by mutations play a role in immunomodulation. However, attenuation cannot be linked to individual mutations or gene disruptions.

  • Structural protein VP2 of African horse sickness virus is not essential for virus replication in vitro [PublishAheadOfPrint]

  • The Reoviridae family consists of non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus and epizootic haemorrhagic disease virus causing notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2 encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 for that of green fluorescence protein (GFP) resulted in AHSV expressing GFP which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced and virus release from insect cells was completely abolished. Further, the other outer capsid protein VP5 was not co-purified with virions for virus mutants without VP2 expression. AHSV without VP5 expression however could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family.

    Importance Members of the Reoviridae family cause major health problems worldwide ranging from lethal diarrhoea by rotavirus in humans to economic losses in livestock production by different orbiviruses. The orbivirus genus contains many virus species of which bluetongue virus, epizootic haemorrhagic disease virus, and African horse sickness virus (AHSV) are causing notifiable diseases according to the World Organisation of Animal Health. Recently, it has been shown that non-structural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1-7 of these non-enveloped architectural complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro. Our findings are very important for virologists working in the field of non-enveloped viruses, in particular reoviruses.

  • LUBAC modulates LMP1 activation of NF{kappa}B and IRF7 [PublishAheadOfPrint]

  • Recently, Linear Ubiquitin Assembly Complex (LUBAC)-mediated linear ubiquitination has come into focus due to its emerging role in activation of NFB in different biological contexts. However, the role of LUBAC in LMP1 signaling leading to NFB and IRF7 activation has not been investigated. We have shown here that RNF31, the key component of LUBAC complex, interacts with LMP1 and IRF7 in EBV-transformed cells, and that LUBAC stimualtes linear ubiquitination of NEMO and IRF7. Consequently, LUBAC is required for LMP1 signaling to full activation of NFB, but inhibits LMP1-stimulated IRF7 transcriptional activity. The protein levels of RNF31 and LMP1 correlate in EBV-transformed cells. Knockdown of RNF31 in EBV-transformed IB4 cells by RNA interference negatively regulates expression of the genes downstream of LMP1 signaling, and results in a decrease of cell proliferation. These lines of evidence indicate that LUBAC-mediated linear ubiquitination play crucial roles in regulating LMP1 signaling and functions.

    Importance We have shown here that LUBAC-mediated linear ubiquitination is required for LMP1 activation of NFB, but inhibits LMP1-mediated IRF7 activation. Our findings provide novel mechanisms underlying EBV-mediated oncogenesis, and may have broad impact on IRF7-mediated immune responses.

  • Mechanism of enhanced HIV restriction by virion coencapsidated cytidine deaminases APOBEC3F and APOBEC3G [PublishAheadOfPrint]

  • The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4+ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine to adenine hypermutation through deamination of cytosine to form uracil in (-)DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity than A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 alone, A3G and A3F when combined had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer, in the absence of RNA, which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded (-)DNA exposed for longer periods of time enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties when compared to each individual counterpart.

    IMPORTANCE The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 viral infectivity factor (Vif) protein. After APOBEC3 enzymes are encaspidated into virions they deaminate cytosines in (-) DNA which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found demonstrating that APOBEC3s still have discernable activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate than an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we may still not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.

  • Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage [PublishAheadOfPrint]

  • Human cytomegalovirus terminase complex cleaves the concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity will be inhibited by known RNase H inhibitors. Using a novel ELISA format screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported as an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point similarly to other published terminase complex inhibitors. Importantly, this inhibitor also prevented cleavage of viral genomic DNA in infected cells. Taken together, the results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.

    IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can result in severe and debilitating consequences. The US Food and Drug Administration approved anti-human cytomegalovirus drugs mainly target the viral polymerase and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.

  • The Norovirus NS3 protein is a dynamic lipid- and microtubule-associated protein involved in viral RNA replication. [PublishAheadOfPrint]

  • Norovirus (NoV) infections are a significant health burden to society yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate dsRNA. We have observed that when expressed individually MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not co-localise with any particular protein markers to cellular organelles, but localised to cellular membranes, in particular those high in cholesterol content. Both proteins also show some degree of co-localisation with the cytoskeleton marker bbeta;-tubulin. Although the distribution of MNV and NV NS3 were similar, NV NS3 displayed a greater level of co-localisation with the Golgi and the ER. However, we observed that although both proteins co-localised in membranes counterstained with filipin, an indicator of cholesterol content, MNV NS3 displayed a greater association with flotillin and stomatin, proteins known to associate with sphingolipid and cholesterol-rich micro-domains. Utilising time-lapse epi-fluorescent microscopy we observed that the membrane derived vesicular structures induced by MNV NS3 were highly motile and dynamic in nature and their movement was dependent on intact microtubules. These results begin to interrogate the functions of NoV proteins during virus replication and highlight the conserved properties of the NoV NS3 protein between the seven Norovirus genogroups.

    IMPORTANCE Many mechanisms involved in the replication of Norovirus still remain unclear including the role for NS3 protein, one of seven non-structural viral proteins, which remains to be elucidated. This study reveals that Murine Norovirus (MNV) NS3 is intimately associated with the viral replication complex and dsRNA. We observed that the NS3 proteins from both MNV and Norwalk virus (NV) induce prominent vesicular structures, and that this formation is dependent on microtubules and cellular cholesterol. Thus this study contributes to our understanding of protein function within different Norovirus genogroups and expands a growing knowledge base on the interaction between positive strand RNA ((+)RNA) viruses and cellular membranes that contribute to biogenesis of viral-induced membrane organelles. This study contributes to our understanding of viral protein function and the ability of a viral protein to recruit specific cellular organelles and lipids which enable replication.

  • Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via Nsp5 by Inducing STAT3 Degradation [PublishAheadOfPrint]

  • Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. Here we report that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV-1 and PRRSV-2 species led to a similar reduction of STAT3 protein level but had minimum effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as STAT3 level decreased along with incremental amount of PRRSV inocula. Further study showed that non-structural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to approximately 3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT signaling, leading to perturbation of the host innate and adaptive immune responses.

    SIGNIFICANCE The typical features of immune responses in PRRSV-infected pigs are delayed onset and low level of virus neutralizing antibodies as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of the protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

  • Alternative polyadenylation of human bocavirus at its 3 end is regulated by multiple elements and affects capsid expression [PublishAheadOfPrint]

  • Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is yet unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site (pA)d, a novel distal polyadenylation site (pA)d2 which is located in the right end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) by sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus and alternative polyadenylation at the HBoV 3rrsquo; end regulates its capsid expression.

    IMPORTANCE The distal polyadenylation site (pA)d of HBoV is located about 400 nts from the right end palindromic terminus, which is different from bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2 does not contain typical polyadenylation signals and the last 42 nts form a stem-loop structure which is almost identical to that of MVC. Further study showed that NS1, REH, and cis elements of (pA)d are required for efficient polyadenylation at (pA)d2. Polyadenylation at (pA)d2 enhances capsid expression. Our study demonstrates alternative polyadenylation at the 3rrsquo; end of HBoV and suggests an additional mechanism by which capsid expression is regulated.

  • Identification of New World Quails Susceptible to ALV-J Infection [PublishAheadOfPrint]

  • The J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na+/H+ exchanger 1. The resistance to J subgroup avian leukosis virus in a great majority of galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na+/H+ exchanger 1. Because there are concerns of transspecies virus transmission, we studied the natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to J subgroup avian leukosis virus infection and replication in vitro and the cloned Na+/H+ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new J subgroup avian leukosis virus variants, but resistant to A and B and weakly susceptible to C and D subgroups of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the J subgroup avian leukosis virus could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential to further evolution.

    IMPORTANCE Since its spread in chicken broilers in China and Southeast Asia in 2000, the ALV-J remains a major enzootic challenge for the poultry industry. Although the virus rapidly diversifies in the poultry, its spill-over and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the virus evolution and its potential host range in wild birds. Because the resistance to avian retroviruses consists particularly in the receptor incompatibility, we studied the Na+/H+ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible for virus entry and we confirmed the susceptibility of four New World quail species in vitro. We propose that a prospective molecular epidemiology study should be conducted to identify the species with the potential to become a reservoir for the ALV-J.

  • The many faces of the flavivirus NS5 protein in antagonism of type I interferon signaling. [PublishAheadOfPrint]

  • The vector-borne flaviviruses cause severe disease in humans on every inhabited continent on earth. Their transmission by arthropods, particularly mosquitoes, facilitates large emergence events such as witnessed with Zika virus (ZIKV) or West Nile virus in the Americas. Every vector-borne flavivirus examined thus far that causes disease in humans, from dengue virus to ZIKV, antagonizes the host type I interferon (IFN-I) response by preventing JAK-STAT signaling, suggesting that suppression of this pathway is an important determinant of infection. The most direct and potent viral inhibitor of this pathway is the nonstructural protein, NS5. However, the mechanism utilized by NS5 from different flaviviruses is often quite different, sometimes despite close evolutionary relationships between viruses. The varied mechanisms of NS5 as an IFN-I antagonist are also surprising given that the evolution of NS5 is restrained by the requirement to maintain function of two enzymatic activities critical for virus replication, the methyltransferase and RNA-dependent RNA polymerase. This review discusses the different strategies used by flavivirus NS5 to evade the antiviral effects of IFN-I and how this information can be used to better model disease and develop antiviral countermeasures.

  • Modeling severe fever with thrombocytopenia syndrome virus infection in golden Syrian hamsters: Importance of STAT2 in preventing disease and effective treatment with favipiravir [PublishAheadOfPrint]

  • Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease endemic in parts of Asia. The etiologic agent, SFTS virus (SFTSV; Bunyaviridae family, Phlebovirus genus) has caused significant morbidity and mortality in China, Korea, and Japan, with key features of disease being intense fever, thrombocytopenia, and leukopenia. Case fatality rates are estimated in the 30% range and no antivirals or vaccines are approved for use for treatment and prevention of SFTS. There is evidence that in human cells, SFTSV sequesters STAT proteins in replication complexes, thereby inhibiting type I interferon signaling. Here, we demonstrate that hamsters devoid of functional STAT2 are highly susceptible to as few as 10 plaque-forming units of SFTSV with animals generally succumbing within 5-6 days after subcutaneous challenge. The disease included marked thrombocytopenia and inflammatory disease characteristic of the human condition. Infectious virus titers were present in the blood and most tissues 3 days after virus challenge and severe inflammatory lesions were found in the spleen and liver samples of SFTSV-infected hamsters. We also show that SFTSV infection in STAT2 knockout (KO) hamsters is responsive to favipiravir treatment protecting all animals from lethal disease and reducing serum and tissue viral loads by 3 to 6 orders of magnitude. Taken together, our results provide additional insights into the pathogenesis of SFTSV infection and support the use of the newly described STAT2 KO hamster model for evaluation of promising antiviral therapies.

    IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease for which there is currently no therapeutic options or available vaccines. The causative agent, SFTS virus (SFTSV), is present in China, Korea, and Japan, and infections requiring medical attention result in death in as high as 30% of cases. Here, we describe a novel model of SFTS in hamsters genetically engineered to be deficient in a protein that helps protect humans and animals against viral infections. These hamsters were found to be susceptible to SFTSV and share disease features associated with the human disease. Importantly, we also show that SFTSV infection in hamsters can be effectively treated with a broad-spectrum antiviral drug approved for use in Japan. Our findings suggest that the new SFTS model will be an excellent resource to better understand SFTSV infection and disease, as well as a valuable tool for evaluating promising antiviral drugs.

  • Porcine reproductive and respiratory syndrome virus nsp1{alpha} inhibits NF-{kappa}B activation by targeting linear ubiquitin chain assembly complex (LUBAC) [PublishAheadOfPrint]

  • Linear ubiquitination, a newly discovered posttranslational modification, is catalyzed by the linear ubiquitin chain assembly complex (LUBAC), which is composed of three subunits: one catalytic subunit HOIP and two accessory molecules, HOIL-1L and SHARPIN. Accumulating evidence suggests that linear ubiquitination plays a crucial role in innate immune signaling and especially in the activation of the NF-B pathway by conjugating linear polyubiquitin chains to NF-B essential modulator (NEMO, also called IKK), the regulatory subunit of the IKK complex. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide, is an ideal model to study the host's disordered inflammatory responses after viral infection. Here, we found that LUBAC-induced NF-B and proinflammatory cytokine expression can be inhibited in the early phase of PRRSV infection. Screening the PRRSV-encoded proteins showed that nonstructural protein 1aalpha; (nsp1aalpha;) suppresses LUBAC-mediated NF-B activation and its CTE domain is required for the inhibition. Mechanistically, nsp1aalpha; binds to HOIP/HOIL-1L and impairs the interaction between HOIP and SHARPIN, thus reducing the LUBAC-dependent linear ubiquitination of NEMO. Moreover, PRRSV infection also blocks LUBAC complex formation and NEMO linear-ubiquitination, the important step for transducing NF-B signaling. This unexpected finding demonstrates a previously unrecognized role of PRRSV nsp1aalpha; in modulating LUBAC signaling, and explains an additional mechanism of immune modulation by PRRSV.

    IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most important veterinary infectious diseases in countries with intensive swine industries. PRRSV infection usually suppresses proinflammatory cytokine expression in the early stage of infection, whereas it induces an inflammatory storm in the late stage. However, precisely how the virus is capable of doing so remains obscure. In this study, we found that by blocking the interaction of its catalytic subunit HOIP and accessory molecule SHARPIN, PRRSV can suppress NF-B signal transduction in the early stage of infection. Our findings not only reveal a novel mechanism evolved by PRRSV to regulate inflammatory responses, but also highlight the important role of linear ubiquitination modification during virus infection.

  • Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain (SRCR5) from Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its world-wide spread in the 1990s. The infection of its causative agent, PRRS virus (PRRSV), is proven to be mediated by an indispensable receptor, porcine CD163 (pCD163) and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for the virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 AAring;. This structure includes a markedly long loop region and shows a special electrostatic potential, which are significantly different from other members in scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently we carried out structure-based mutational studies to identify the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during the virus invasion. Altogether the current work provides the first view of CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus.

    IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses to pig farming. The syndrome is caused by the PRRS virus (PRRSV) and the infection of PRRSV is shown to be mediated by the host cell surface receptors. One of them, porcine CD163 (pCD163), is especially indispensable and its fifth scavenger receptor cysteine-rich domain (SRCR5) has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides the structural information of CD163 SRCR domain for the first time, but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

  • Infectious Bursal Disease Virus activates c-Src to promote {alpha}4{beta}1 integrin-dependent viral entry via modulating downstream Akt-RhoA GTPase-actin rearrangement cascade [PublishAheadOfPrint]

  • While the entry of IBDV is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after the receptor binding and how they contribute to the viral entry remain elusive. We show here that IBDV activates c-Src by inducing phosphorylation of the Y416 residue in c-Src both in the DF-1 chicken fibroblasts and in vivo in the bursa of fabricius from SPF chickens. Importantly, the inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces much higher level of c-Src Y416 phosphorylation than an attenuated strain. Inhibition of c-Src activation by Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on the virus adhesion. Furthermore, shRNA downregulation of integrin either aalpha;4 or bbeta;1 subunit but not HSP90 remarkably attenuates the IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in the IBDV internalization but not the virus adhesion. Moreover, interestingly, inhibition of c-Src downstream either PI3K/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV induced high levels of c-Src phosphorylation. Cumulatively our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting itself cell entry via an integrin-mediated pathway by activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement.

    IMPORTANCE While the IBDV caused immunosuppression is highly related to the viral invasion, the molecular basis of IBDV cellular entry remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing phosphorylation of the Y416 residue in c-Src to promote viral internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with its pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is aalpha;4bbeta;1 integrin but not HSP90-dependent, and involves activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential c-Src as candidate target for the development of the IBDV therapeutic drugs.

  • Activation and Inactivation of Primary Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Trimers by CD4-Mimetic Compounds [PublishAheadOfPrint]

  • Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that activation/inactivation represents an antiviral mechanism common to all tested CD4mc. Biphasic dose-response curves indicate that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on-rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility.

    IMPORTANCE The trimeric envelope glycoproteins of the human immunodeficiency virus (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, trigger changes in the conformation of the HIV-1 envelope glycoprotein trimer important for virus entry. Small-molecule CD4-mimetic compounds inhibit HIV-1 infection by multiple mechanisms: 1) Direct blockade of the interaction between the gp120 exterior envelope glycoprotein and CD4; 2) Premature triggering of conformational changes in the envelope glycoproteins, leading to irreversible inactivation; and 3) Exposure of cryptic epitopes to antibodies, allowing virus neutralization. The consequences of the binding of the CD4-mimetic compound to the HIV-1 envelope glycoproteins depends upon how many of the three subunits of the trimer are bound and upon the propensity of the envelope glycoproteins to undergo conformational changes. Understanding the mechanistic factors that influence the activity of CD4-mimetic compounds can help to improve their potency and coverage of diverse HIV-1 strains.

  • Phylogenetic diversity of Koala Retrovirus within a Wild Koala Population [PublishAheadOfPrint]

  • Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala Retrovirus (KoRV) is a gammaretrovirus present in most wild Koala populations and captive colonies. Five subtypes of KoRV have been identified (A-E) based on amino acid sequence divergence in a hyper-variable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has primarily been conducted on KoRV in captive koalas housed in zoos in Japan, the United States of America and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from south-east Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into tree major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hyper-variable region subtypes (C, D and E) as well as four hyper-variable region subtypes that we newly define here (F, G, H and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D and F were found to be at high prevalence, while subtypes G, H and I were present in a smaller number of animals.

    IMPORTANCE Koala Retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence amongst a wild koala population and significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ-line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

  • Inclusion body fusion of human parainfluenza virus type 3 regulated by acetylated {alpha}-tubulin enhances viral replication [PublishAheadOfPrint]

  • Viral inclusion bodies (IBs) or replication factories are unique structures generated by viral proteins together with some cellular proteins as a platform for efficient viral replication, but little is known about the mechanism underlying IB formation and fusion. Our previous study demonstrated that the interaction between the nucleoprotein (N) and phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3), an enveloped virus of great medical impact, can form IBs. In this study, we found that small IBs can fuse with each other to form large IBs that enhance viral replication. Furthermore, we found that acetylated aalpha;-tubulin interacts with the N-P complex and colocalizes with IBs of HPIV3 but does not interact with the N-P complex of human respiratory syncytial virus or vesicular stomatitis virus and does not colocalize with IBs of human respiratory syncytial virus. Most importantly, enhancement of aalpha;-tubulin acetylation using the pharmacological inhibitor TSA, RNAi knockdown of the deacetylase enzymes HDAC6 and SIRT2, or by the expression of aalpha;-TAT1 resulted in the fusion of small IBs into large IBs and effective viral replication. In contrast, suppression of acetylation of aalpha;-tubulin by over-expressing HDAC6 and SIRT2 profoundly inhibited the fusion of small IBs and viral replication. Our findings offer previously unidentified mechanistic insights into the regulation of viral IB fusion by acetylated aalpha;-tubulin, which is critical for viral replication.

    IMPORTANCE Inclusion bodies (IBs) are unique structures generated by viral proteins and some cellular proteins as a platform for efficient viral replication. Human parainfluenza virus type 3 (HPIV3) is a nonsegmented single-stranded RNA virus that mainly causes lower respiratory disease in infants and young children. However, no vaccines or antiviral drugs for HPIV3 are available. Therefore, understanding virus-host interactions and developing new antiviral strategies are increasingly important. Acetylation on lysine (K) 40 of aalpha;-tubulin is an evolutionarily conserved modification and plays an important role in many cellular processes, but its role in viral IB dynamics has not been fully explored. To our knowledge, our findings are the first to show that acetylated aalpha;-tubulin enhances viral replication by regulating HPIV3 IB fusion.

  • Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production [PublishAheadOfPrint]

  • Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product prostaglandin E2 (PGE2) are known to play important roles in the modulation of both the host response to the infection and replicative cycle of several viruses. However, the precise mechanism(s) by which the COXs/PGE2 pathway regulates sapoviruses replication remains largely unknown. In this study, the infection of porcine sapovirus (PSaV) Cowden strain, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 hours post-infection (hpi) with only a transient increase of COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors such as nonsteroidal anti-inflammatory drugs or siRNAs against COX-1 and COX-2 significantly reduced PGE2 production as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with the activation of the COXs/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-L-Methyl-Arginine-ester. This study has identified pivotal role for the COXs/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of these poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection.

    IMPORTANCE Sapoviruses are one of major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) Cowden strain, we demonstrate that PSaV induced the vitalization of cyclooxygenases (COXs) and prostaglandin E2 (PGE2) pathway. Targeting of COX1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2 specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with the activation of the COXs/PGE2 pathway. We further demonstrate that the production of PGE2 provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be new targets for treatment of sapovirus infection.

  • Suppression of Adenovirus Replication by Cardiotonic Steroids [PublishAheadOfPrint]

  • The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome, potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yield of four adenoviruses (HAdV-A31, B35, C5 and a species D conjunctivitis isolate) by at least 2- 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein, hexon, are compromised. qRT-PCR analyses revealed that both drugs altered E1A RNA splicing (favouring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on continued presence of the drug and rapidly reversible. RIDK34, a derivative of convallotoxin, although, having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin/digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

    IMPORTANCE Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

  • Glycosyl-phosphatidylinositol (GPI)-anchored anti-HIV scFv efficiently protect CD4 T cells from HIV-1 infection and deletion in hu-PBL mice [PublishAheadOfPrint]

  • Despite the success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from homozygous CCR5 32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance of permissive cells. Here, we employed a glycosyl-phosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing the GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)- and dual tropic- HIV-1 and had survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after co-transfused with HIV-infected cells, significantly reduced viral loads and viral RNA copies relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as genetic intervention against both R5- and X4-tropic HIV-1 infection.

    IMPORTANCE Blocking HIV-1 virus entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 co-receptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, emergence of viruses using CXCR4 co-receptor is a concern for therapies applying single co-receptor disruption. We here report that HIV-1 permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4- or dual tropic virus infection ex vivo. In a pre-clinical study using hu-PBL mice, we show CD4 T cells were protected and GPI-scFv X5-transduced cells were selected in HIV-1 infected animals. Moreover, we show that GPI-scFv X5-transduced CD4 T cells exerted negative effect on virus replication in vivo. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as genetic intervention against both R5- and X4-tropic HIV-1 infection.

  • MicroRNA-373 Facilitated the Replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by Its Negative Regulation of Type I Interferon Induction [PublishAheadOfPrint]

  • microRNAs (miRNAs) played an important role in the regulation of immune response. Previous studies have indicated that dysregulating the miRNAs may lead to the immunosuppression of porcine reproductive and respiratory syndrome virus (PRRSV). However, it's not clear about how PRRSV regulated the expression of host miRNA which may make the immune escape or promote the replication of the virus. The present work suggested that PRRSV up-regulated the expression of miR-373 through elevating the expression of specificity protein 1 (Sp1) in MARC-145 cells. Furthermore, this work demonstrated that miR-373 promoted the replication of PRRSV since miR-373 was a novel negative miRNA to the production of interferon (IFN)-bbeta;by targeting nuclear factor I (NFI) A, NFIB, interleukin-1 receptor-associated kinase (IRAK)1, IRAK4 and interferon regulatory factor (IRF) 1. This paper also found that both NFIA and NFIB were the novel proteins for inducing the production of IFN-bbeta;and both of them could inhibit the replication of PRRSV. In conclusion, PRRSV up-regulated the expression of miR-373 by elevating the expression of Sp1 and hijacked the host miR-373 to promote the replication of PRRSV by negatively regulating the production of IFN-bbeta;.

    Importance PRRSV caused one of the most economically devastating diseases of swine and there's no effective method for controlling PRRSV. It's not clear about how PRRSV inhibited the host's immune response and induced the persistent infection. Previous studies have shown that PRRSV inhibited the production of type I IFN and the treatment of type I IFN could efficiently inhibit the replication of PRRSV, so it will be helpful to design new methods of controlling PRRSV by understanding the molecular mechanism that PRRSV modulated the production of IFN. The current work showed that the miR-373 up-regulated by PRRSV promoted PRRSV replication since miR-373 impaired the production of IFN-bbeta;by targeting NFIA, NFIB, IRAK1, IRAK4 and IRF1. And both NFIA and NFIB were the antiviral proteins to PRRSV. In conclusion, this paper revealed a novel mechanism that PRRSV impaired the production of type I IFN by up-regulating miR-373 expression in MARC-145 cells.

  • Dual function of the pUL7-pUL51 tegument protein complex in HSV-1 infection [PublishAheadOfPrint]

  • The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus-1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other and that they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore we have also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected calls, and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to its surroundings through modulating the activity of focal adhesion complexes.

    IMPORTANCE The Herpesviridae is a large family of highly-successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus-1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host-cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells.

  • ZIKA virus efficiently replicates in human retinal pigment epithelium and disturbs its permeability [PublishAheadOfPrint]

  • Recently, the Flavivirus Zika virus (ZIKV) has rapidly spread in the Americas and in the Caribbean islands....

  • NES masking regulates HIV-1 Rev trafficking and viral RNA nuclear export [PublishAheadOfPrint]

  • HIV-1's Rev protein forms a homooligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study we used a functional fluorescently-tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found Rev activity to be remarkably tolerant of diverse NES sequences including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position- and multimerization-dependent, consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.

    IMPORTANCE HIV-1 infects more than 34 million people worldwide causing ggt;1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal (NLS) and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. Here we provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities; both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

  • Robust CNS pathology in transgenic mice following peripheral injection of {alpha}-synuclein fibrils [PublishAheadOfPrint]

  • Misfolded aalpha;-synuclein (aalpha;S) is hypothesized to spread throughout the CNS by neuronal connectivity leading to widespread pathology. Increasing evidence indicates that it also has the potential to invade the CNS via peripheral nerves in a prion-like manner. Based on the effectiveness following peripheral routes of prion administration, we extend our previous studies of CNS neuroinvasion in M83 aalpha;S transgenic mice following hindlimb muscle (IM) injection of aalpha;S fibrils by comparing various peripheral sites of inoculations with different aalpha;S protein preparations. Following intravenous injection in the tail vein of M83+/+ mice, robust aalpha;S pathology was observed in the CNS without the development of motor impairments within the time frame examined. Intraperitoneal (IP) injections of aalpha;S fibrils in M83+/- mice resulted in CNS aalpha;S pathology associated with paralysis. Interestingly, injection with soluble, non-aggregated aalpha;S resulted in paralysis and pathology in only a subset of mice, whereas soluble 71-82 aalpha;S, human bbeta;S, and KLH control proteins induced no symptoms or pathology. IP injection of aalpha;S fibrils also induced CNS aalpha;S pathology in another aalpha;S transgenic mouse line (M20), albeit less robust in these mice. In comparison, IM injection of aalpha;S fibrils was more efficient in inducing CNS aalpha;S pathology in M83 mice than IP or tail vein injections. Furthermore, IM injection of soluble, non-aggregated aalpha;S in M83+/- mice also induced paralysis and CNS aalpha;S pathology, although less efficiently. These results further demonstrate the prion-like characteristics of aalpha;S and reveal its efficiency to invade the CNS via multiple routes of peripheral administration.

    IMPORTANCE The misfolding and accumulation of aalpha;-synuclein (aalpha;S) inclusions is found in a number of neurodegenerative disorders and is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Similar characteristics have been observed between the infectious prion protein and aalpha;S, including its ability to spread from the peripheral nervous system and along neuroanatomical tracts within the central nervous system. In this study, we extend our previous results and investigate the efficiency of intravenous (IV), intraperitoneal (IP), and intramuscular (IM) routes of injection of aalpha;S fibrils and other protein controls. Our data reveals that injection of aalpha;S fibrils via these peripheral routes in aalpha;S overexpressing mice are capable of inducing a robust aalpha;S pathology and in some cases cause paralysis. Furthermore, soluble, non-aggregated aalpha;S also induced aalpha;S pathology, albeit with much less efficiency. These findings further support and extend the idea of aalpha;S neuroinvation from peripheral exposures.

  • Age dependent differences in pseudorabies virus neuropathogenesis and associated cytokine expression. [PublishAheadOfPrint]

  • Severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related with the age of the pig. During this study, pigs of 2- and 15-week-old were inoculated with the PRV strain NIA3. This resulted in important clinical disease although associated morbidity and mortality were lower in older pigs. qPCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence however sorted from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was found in the nasal mucosa, suggesting that other sites may serve as primary replication site. Secondly, PRV reached the trigeminal ganglion (TG) and brainstem rapidly upon infection, but compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs and the decrease of viral mRNA expression was not preceded or associated with an increased cytokine expression. Thirdly, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulb of pigs of both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV induced clinical signs are likely due to enhanced viral replication and associated immunopathology in immature TG and central nervous system (CNS) neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb.

    IMPORTANCE It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in new-borns compared to older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms remain however unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response, but is more likely to reflect the immature development state of TG and CNS neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

  • KSHV microRNAs Target GADD45B to Protect Infected Cells from Cell Cycle Arrest and Apoptosis [PublishAheadOfPrint]

  • Kaposi's sarcoma is one of the most common malignancies in HIV-infected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8) expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs may target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and that KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen with targeting GADD45B with siRNAs as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly ADP ribose polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses.

    Importance Kaposi's sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense mechanisms designed to eradicate the viral infection. One such response can include DNA damage response factors, which can promote an arrest in cell growth and trigger cell death. We used a new approach to search for human genes repressed by small nucleic acids (microRNAs) expressed by a gammaherpesvirus (KSHV), which identified a gene called GADD45B as a target of microRNAs. Repression of GADD45B, which is expressed in response to DNA damage, benefited survival of infected cells in response to a DNA damage response. This information could be used to design new treatments for herpesvirus infections.

  • Palmitoylation of Sindbis virus TF protein regulates its plasma membrane localization and subsequent incorporation into virions [PublishAheadOfPrint]

  • Palmitoylation is a reversible, post-translational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms identical in the N-terminus but unique in the C-terminus due to a (-1) ribosomal frameshift during translation. In this study we used cysteine (Cys) mutants to test differential palmitoylation of Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N-terminus of 6K and TF, the four additional Cys residues in TF's unique C-terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation primarily occurs in the N-terminus. In contrast, 6K is not palmitoylated even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a higher incidence of abnormal particle morphologies compared to wild type virus by transmission electron microscopy. We propose a model where the C-terminus of TF modulates the palmitoylation of TF at the N-terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding.

    IMPORTANCE Alphaviruses are a re-emerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions and optimal levels of TF correlate positively with wild type-like particle morphology. In this study we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants where TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigating the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

  • Adeno-Associated Virus Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5 and 11 [PublishAheadOfPrint]

  • Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown AAP to be essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among the AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to 12 (i.e., AAPs derived from AAV1 to 12, respectively) and examined AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopies, Barcode-Seq and quantitative dot blot assay. The study revealed that AAP1 to 12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, 5, 11 and 12; assembled AAV5, 8 and 9 capsids are excluded from the nucleolus, in contrast to nucleolar enrichment of assembled AAV2 capsids; and surprisingly, AAV4, 5 and 11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process, and challenge the current notions about the role of AAP and the nucleolus in capsid assembly.

    IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) show that assembly takes place in the nucleolus, is dependent on AAP, and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to 12), we find that AAP is not an essential requirement for capsid assembly of AAV4, 5 and 11, and AAP, assembled capsids and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly, and show the heterogeneous nature of the assembly process within the AAV family.

  • A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells [PublishAheadOfPrint]

  • Rabies still remains a public health threat in most parts of the world, and approximately 99% of the cases are transmitted by dogs. There is an urgent need to develop an efficacious and affordable vaccine to control canine-transmitted rabies in developing countries. Our previous studies demonstrate that overexpression of chemokines/cytokines such as CCL-3 (MIP-1aalpha;) and GM-CSF can enhance the immunogenicity of rabies vaccines. In the present study, the chemokine CXCL13 was inserted into the genome of the recombinant rabies virus (rRABV) strain LBNSE, and the effect of the chemokine CXCL13 on the immunogenicity of the RABV was investigated. It was found that LBNSE-CXCL13 recruited follicular helper T (Tfh) and germinal center (GC) B cells, promoted the formation of GCs, and increased the population of plasma cells in immunized mice. Further studies showed that mice immunized with LBNSE-CXCL13 produced more rabies virus-neutralizing antibodies (VNAs) and developed better protection than those immunized with the parent virus LBNSE or the GM-CSF-expressing RABV (LBNSE-GM-CSF). Collectively, these findings provide a better understanding of the role of CXCL13 expression in the immunogenicity of the RABV, which may help in designing more efficacious rabies vaccines.

    Importance Rabies is endemic in most parts of the world, and more effort is needed to develop affordable and effective vaccines to control or eliminate this disease. The chemokine CXCL13 recruits both Tfh and B cells, which is essential for the homing of Tfh cells and the development of B cell follicles. In this study, the effect of the overexpression of CXCL13 on the immunogenicity of the RABV was evaluated in a mouse model. We found that CXCL13 expression promoted humoral immunity by recruiting Tfh and GC B cells, facilitating the formation of GCs and increasing the number of plasma cells. As expected, the overexpression of CXCL13 resulted in enhanced virus-neutralizing antibody (VNA) production and protection against a virulent RABV challenge. These findings provide a better understanding of the role of CXCL13 in RABV-induced immune responses, which will help in designing more efficacious rabies vaccines.

  • Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins [PublishAheadOfPrint]

  • The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells, but did not neutralize virus, and bound only moderately to the same cells that they killed so effectively as ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities, and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection.

    IMPORTANCE There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells, and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective anti-retroviral therapy.

  • The Potential for Low Pathogenic Avian H7 Influenza A Viruses to Replicate and Cause Disease in a Mammalian Model [PublishAheadOfPrint]

  • H7 subtype influenza A viruses are widely distributed and have been responsible for human infections and numerous outbreaks in poultry with significant impact. Despite this, the disease-causing potential of the precursor low pathogenic (LP) H7 viruses from the wild bird reservoir has not been investigated. Our objective was to assess the disease causing potential of 30 LP H7 viruses isolated from wild avian species in the USA and Canada, using the DBA/2J mouse model. Without prior mammalian adaptation, the majority of viruses, 27 (90%), caused mortality in mice. Of these, 17 (56.7%) caused 100% mortality and 24 were of similar pathogenicity to A/Anhui/1/2013 (H7N9), which is highly pathogenic in mice. Viruses of duck-origin were more pathogenic than those of shorebird-origin, as 13 of 18 (72.2%) duck-origin viruses caused 100% mortality whilst 4 of 12 (33.3%) shorebird-origin viruses caused 100% mortality, despite there being no difference in mean lung viral titers between these groups. Replication beyond the respiratory tract was also evident, particularly in the heart and brain. Of the 16 viruses studied for fecal shedding, 11 were detected in fecal samples. These viruses exhibited a strong preference for avian-type aalpha;2,3-linked sialic acids, however, binding to mammalian-type aalpha;2,6-linked sialic acids was also detected. These findings indicate that LP avian H7 influenza A viruses are able to infect and cause disease in mammals without prior adaptation and therefore pose a potential public health risk.

    IMPORTANCE Low pathogenic (LP) avian H7 influenza A viruses are widely distributed in the avian reservoir and are the precursors of numerous outbreaks of highly pathogenic avian influenza viruses in commercial poultry farms. However, unlike highly pathogenic H7 viruses, the disease-causing potential of LP H7 viruses from the wild bird reservoir has not been investigated. To address this we studied 30 LP avian H7 viruses isolated from wild avian species in the USA and Canada using the DBA/2J mouse model. Surprisingly, the majority of these viruses, 90%, caused mortality in mice without prior mammalian adaptation and 56.7% caused 100% mortality. There was also evidence of spread beyond the respiratory tract and fecal shedding. Therefore, the disease-causing potential of LP avian H7 influenza A viruses in mammals may be underestimated and these viruses therefore pose a potential public health risk.

  • Honey bee deformed wing virus structures reveal conformational changes accompany genome release [PublishAheadOfPrint]

  • The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the 3-D structures of DWV capsids isolated from infected honey bees, including the immature procapsid, genome-filled virion, putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the five-fold vertices. For the procapsid and genome filled capsids the five-fold spikes had an open flower-like conformation whereas the putative A-particle and empty capsids that have released the genome had a closed tube-like spike conformation. Between the two conformations the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that genome may escape from a five-fold vertex to initiate infection. Finally the structures illustrate that similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release.

    Significance: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allow the first glimpse into conformational changes that may be required during transmission and genome release for DWV.

  • Mapping of Functional Subdomains in Terminal Protein Domain of Hepatitis B Virus Polymerase [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) encodes a multi-function reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related Hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP whose sequences are highly conserved. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a "priming loop" in TP, which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming, but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures, and will facilitate antiviral targeting of this unique domain.

    Importance Hepatitis B virus (HBV) is a major cause of viral hepatitis, liver cirrhosis and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the terminal protein (TP) domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structuremmdash;where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

  • A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling [PublishAheadOfPrint]

  • Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. PP2A, YAP, Src family tyrosine kinases, Shc, phosphoinositide-3-kinase (PI3K) and PLC1 have all been implicated in MT transformation. Mutant dl1015, deleting residues 338-347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets SH3 domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation-defective. Extensive analysis of E349K is described here. Similar to wild type MT, E349K associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase and PLC1. E349K was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by co-immunoprecipitation showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of E349K with PI3K, cells expressing the mutant failed to elevate PIP3 in mutant expressing cells. These results indicate a novel unsuspected aspect to PI3K control.

    IMPORTANCE Middle T (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

  • Ablation of programmed -1 ribosomal frameshifting in Venezuelan equine encephalitis virus results in attenuated neuropathogenicity [PublishAheadOfPrint]

  • The Alphaviruses, Venezuelan, eastern and western equine encephalitis viruses (VEEV, EEEV, WEEV) are arthropod-borne (+) RNA viruses that are capable of causing acute and fatal encephalitis in many mammals including humans. VEEV was weaponized during the Cold War, and is recognized as a select agent. Currently, there are no FDA approved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscore the need for research aimed at developing medical countermeasures. These viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the viral trans-frame protein (TF), which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the Alphavirus -1 PRF signals were characterized revealing novel -1 PRF stimulatory structures. -1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells, but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggests that the Alphavirus TF protein is important for passage through the blood-brain barrier, and/or for neuroinvasiveness. These findings suggest a novel approach toward development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related -1 PRF utilizing viruses.

    IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is as a select agent that has been weaponized. This arthropod-borne (+) RNA virus causes acute and fatal encephalitis in many mammals including humans. There is no vaccine or other approved therapeutic. VEEV and related Alphaviruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the viral trans-frame protein (TF), which is important for neuropathogenesis. -1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that TF protein is critical for neurological disease. These findings suggest a new approach toward the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

  • Visualizing the essential role of complete virion assembly machinery in efficient hepatitis C virus cell-to-cell transmission by viral infection activated split-intein mediated reporter system (VISI) [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infects 2-3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology, antiviral drug development and drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, HCV-dependent fluorescence relocalization (HDFR) system allows live-cell visualization of infection without modifying viral genome, but this strategy required careful recognition of fluorescence relocalization pattern for its high fluorescence background in cytoplasm. In this study, to achieve background free visualization of HCV infection, a viral infection activated split-intein mediated reporter system (VISI) was devised. Uninfected huh7.5.1-VISI cell shows no background signal, while HCV infection specifically illuminates the nuclei of infected Huh7.5.1-VISI cells with either GFP or mCherry. Combining VISI-GFP and VISI-mCherry systems, we revisited HCV cell-to-cell transmission with clear-cut distinction of donor and recipient cells in live-cell manner. Independent of virion assembly, exosomes have been reported to transfer HCV sub-genomic RNA to initiate replication in uninfected cells, which suggested an assembly free pathway. However, our data demonstrated that HCV structural genes and p7 gene were essential for not only cell-free infectivity but also cell-to-cell transmission. Additionally, depletion of apoE from donor cells but not from recipient cells significantly reduced HCV cell-to-cell transmission efficiency. In summary, we developed a background-free cell-based reporter system for convenient live-cell visualization of HCV infection and our data indicate that complete HCV virion assembly machinery is essential for both cell-free and cell-to-cell transmission.

    IMPORTANCE Hepatitis C virus (HCV) infects hepatocytes via two pathways: cell-free infection and cell-to-cell transmission. Structural module of HCV genome are required for production of infectious cell-free virions, however role of specific genes within structural module in cell-to-cell transmission is not clearly defined. Our data demonstrate that deletion of core, E1E2 and p7 gene individually results in no HCV cell-to-cell transmission and apoE knockdown from donor cells causes less efficient cell-to-cell transmission. Thus this work indicates complete HCV assembly machinery is required for HCV cell-to-cell transmission. At last this work presents an optimized viral infection activated split-intein mediated reporter system for easy live-cell monitoring of HCV infection.

  • Hepatitis C Virus-Induced Rab32 Aggregation and its Implication in Virion Assembly [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were up-regulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted predominantly expressed GTP- to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core and deposited core into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly.

    IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at transcriptional level and altered the balance between GDP- and GTP-bound Rab32 towards production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core and enriched core into the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.

  • CryoEM structures of expanded poliovirus with VHHs sample the conformational repertoire of the expanded state. [PublishAheadOfPrint]

  • Using cryoelectron microscopy, expanded 80S-like poliovirions were visualized in complexes with four 80S-specific camelid VHHs (Nanobodiesrreg;). In all four complexes, the VHHs bind to a site on the top surface of capsid protein VP3, which is hidden in native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the nanobodies has sampled a range of low energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding has permitted a more detailed view of the virus structure than previously was possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered, and which become rearranged. The higher resolution of these structures has also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, that in retrospect were present in lower-resolution structures but not recognized. These structural observations help to explain pre-existing mutational data, and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion.

    IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection, and how these changes in the virus lead to productive infection events.

  • Pre-mRNA processing factor Prp18 is a stimulatory factor of influenza virus RNA synthesis and possesses the NP chaperone activity [PublishAheadOfPrint]

  • The genome of influenza virus (vRNA) is associated with nucleoprotein (NP) and viral RNA-dependent RNA polymerases, and forms helical viral ribonucleoprotein (vRNP) complexes. NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using an yeast replicon system and single-gene deletion library of yeast (Naito T, Kiyasu Y, Sugiyama K, Kimura A, Nakano R, Matsukage A, Nagata K. Proc. Natl. Acad. Sci., USA, 104:18235-18240, 2007). In infected Prp18 knock-down (KD) cells, synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate the in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from template after early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates the influenza viral RNA synthesis as an NP chaperone for the processive elongation reaction.

    IMPORTANCE Templates for viral RNA synthesis of negative-stranded RNA viruses are not naked RNA but rather RNA encapsidated by viral nucleocapsid proteins forming vRNP complexes. However, viral basic proteins tend to aggregate under the physiological ionic strength without chaperones. We identified pre-mRNA processing factor Prp18 as a stimulatory factor for influenza virus RNA synthesis. We found that one of targets of Prp18 is NP. Prp18 facilitates the elongation reaction of viral polymerases by preventing the deleterious annealing of newly synthesized RNA to template. Prp18 functions as a chaperone for NP to stimulate the formation of NP-RNA complexes. Based on these results, we would propose that Prp18 may be required to maintain the structural integrity of vRNP for processive template reading.

  • Coat protein regulation by CK2, CPIP, HSP70 and CHIP is required for Potato virus A replication and coat protein accumulation [PublishAheadOfPrint]

  • We demonstrate here that coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat-shock proteins, CP-interacting protein (CPIP) and HSP70, are both essential for Potato virus A (PVA; genus Potyvirus) replication and that all of these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Thr243 residue within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation mimicking Asp (CPADA) or phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of non-replicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased PVA translation. From our previous studies we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, this suggest that binding by non-phosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperon activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication.

    IMPORTANCE Host protein kinase CK2, two host chaperones CPIP and HSP70 and viral coat protein (CP) phosphorylation at Thr243 are needed for Potato virus A (PVA) replication. Our results show that non-phosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3rrsquo; end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70.

  • Enterovirus 71 3C promotes apoptosis through cleavage of PinX1, a telomere binding protein [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is an emerging pathogen causing hand, foot and mouth disease (HFMD) and fatal neurological diseases in infants and young children due to their under-developed immune-competence. EV71 infection can induce cellular apoptosis through a variety of pathways that promotes EV71 release. The viral protease 3C plays an important role in EV71-induced apoptosis. However, the molecular mechanism responsible for 3C-triggered apoptosis remains elusive. Here, we found that EV71 3C directly interacted with PinX1, a telomere binding protein. Furthermore, 3C cleaved PinX1 at the site of Q50-G51 pair through its protease activity. Overexpression of PinX1 reduced the level of EV71-induced apoptosis and EV71 release, whereas depletion of PinX1 by small interfering RNA promoted apoptosis induced by etoposide and increased EV71 release. Taken together, our study uncovered a mechanism EV71 utilized to promote host cell apoptosis through cleaving cellular protein PinX1 by 3C.

    IMPORTANCE EV71 3C plays an important role in processing viral proteins and interacting with host cells. In this study, we showed that 3C promoted apoptosis through cleaving PinX1, a telomere binding protein, and this cleavage facilitated EV71 release. Our study demonstrated that PinX1 played an important role in EV71 release and revealed a novel mechanism EV71 utilized to induce apoptosis. These finding is important in understanding EV71-host cell interactions and has potential impact on understanding other Enterovirus-host cell interactions.

  • Mutations Inactivating Herpes Simplex Virus-1 miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia [PublishAheadOfPrint]

  • Herpes simplex virus-1 (HSV-1) latency entails repression of "lytic" gene expression. An attractive hypothesis to explain some of this repression involves inhibition of expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 into HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2, but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently-infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. This latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency.

    IMPORTANCE Herpes simplex virus-1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain repression of "lytic" gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2 deficient mutant virus, we found no evidence that miR-H2 represses expression of ICP0 or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress ICP0 expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for repression of lytic gene expression during latency deserve investigation.

  • Domain interaction studies of HSV-1 tegument protein UL16 reveal its interaction with mitochondria [PublishAheadOfPrint]

  • The UL16 tegument protein of herpes simplex virus type-1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pair-wise transfections, UL16-binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and CTD. Here, we report that the separated and co-expressed functional domains of UL16 are mutually responsive to each other in transfected cells, and form complexes that are stable enough to be captured in co-immunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined.

    IMPORTANCE HSV-1 encoded tegument protein UL16 is involved in multiple events of virus replication cycle ranging from virus assembly to cell-cell spread of the virus, and hence can serve as an important drug target. Unfortunately, lack of both the structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just HSV-1 UL16, but also other herpesviruses, all of which contain a homolog of UL16.

  • Functional characterization of adaptive mutations during the West African Ebola virus outbreak [PublishAheadOfPrint]

  • The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: A substitution D759G in the active center of the polymerase L, a substitution A82V in the receptor-binding domain of the surface glycoprotein GP and a substitution R111C in the self-assembly domain of the RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study shows that virus variants with improved fitness emerged early during the West African EBOV outbreak.

    IMPORTANCE The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral polymerase L, the surface glycoprotein GP and the nucleocapsid protein NP emerged, were fixed early in the outbreak and found in almost 90% of the sequences. Here we showed that these mutations affected functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on potential role of these changes in virus transmissibility and pathogenicity.

  • Parvovirus capsid structures required for infection: mutations controlling receptor recognition and protease cleavages [PublishAheadOfPrint]

  • Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the ssDNA genome to the nucleus where viral replication occurs. Here, we have examined capsid substitutions that eliminate canine parvovirus (CPV) infectivity, and have identified how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to the transferrin receptor type 1 (TfR), particularly during virus disassociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by ggt;100-fold, and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ~10% of the capsid proteins which also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids.

    IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways, and how those contribute to viral infectivity.

  • Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44highCD62LlowCD8+ TEM Cells and Protect "Humanized" HLA-A*02:01 Transgenic Mice Against Ocular Herpes [PublishAheadOfPrint]

  • The herpes simplex virus type 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the "natural protection" seen in asymptomatic (ASYMP) individuals (have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 693 amino acids sequence of VP13/14 protein. Three out of ten epitopes exhibited high to moderate binding affinity to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*0201 positive ASYMP vs. symptomatic (SYMP) individuals (who have frequent clinical herpetic diseases), using a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-, TNF-aalpha; and IL2 production. Higher frequency of multi-functional CD8+ T cells directed against three epitopes, VP13/14286-294, VP13/14504-512 and VP13/14544-552, was predominantly detected inASYMP compared to SYMP individuals. The three epitopes also predominantly recalled more CD45RAlowCD44highCCR7lowCD62LlowCD8+ effector memory T cells (TEM) in ASYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three "ASYMP" CD8+ TEM cell epitopes induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ Tcells that should guide the development of a safe and effective T-cell-based herpes vaccine.

    IMPORTANCE Although most herpes simplex virus 1 (HSV-1) infected individuals shed the virus in their body fluids, following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that, compared to SYMP patients, the "naturally protected" ASYMP individuals have a higher frequency of effector memory CD8+ T cells (CD8+ TEM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14: (VP13/14286-294, VP13/14504-512 and VP13/14544-552). Moreover, immunization of "humanized" HLA-A*02:01 transgenic mice with the three "ASYMP" CD8+ TEM cell epitopes induced robust and polyfunctional HSV-specific CD8+ Tcells associated with a strong protective immunity against ocular herpes infection and disease. The findings support the emerging concept of developing a safe and effective asymptomatic herpes simplex vaccine that is selectively based on "ASYMP" CD8+ T cell epitopes.

  • PrP knockout cells expressing transmembrane PrP resist prion infection [PublishAheadOfPrint]

  • Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a non-raft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that non-raft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC. To further explore these questions, constructs containing either a C terminal wild type GPI anchor signal sequence or a non-raft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying TSE infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e. membrane-bound as in brain microsomes from wild type mice, or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion.

    IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC, or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres. We show that only GPI-anchored PrPC was able to convert to PrPres, and able to serially propagate. The results strongly suggest that GPI anchoring and the localisation of PrPC to rafts is crucial to the ability of PrPC to propagate as a prion.

  • Visualization of HIV T cell virological synapses and virus-containing compartments by 3D correlative light and electron microscopy [PublishAheadOfPrint]

  • Virological synapses (VS) are adhesive structures that form between infected and uninfected cells that can enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM) we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles, and large fluorescent signals correlated with membranous Gag-containing structures, with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and non-endocytic compartments in VS target T cells.

    Importance This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bonafide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses.

  • Substitution of porcine CD163 SRCR domain 5 with a CD163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome (PRRS) viruses. [PublishAheadOfPrint]

  • CD163 knockout (KO) pigs are resistant to infection with genotype 2 (Type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to Type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both Type 1 and Type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knock out (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletions did not express CD163. When place in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six Type 1 and nine Type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all Type 2 isolates, but no Type 1 viruses. Infection of CD163-modified pigs with representative Type 1 and Type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though Type 1 and Type 2 viruses are considered phenotypically similar at several levels, there exists a distinct difference between the viral genotypes in the recognition of CD163.

    Importance Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS; the most costly viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

  • Crystal Structure of the Marburg Virus VP35 Oligomerization Domain [PublishAheadOfPrint]

  • Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a distinct genus from Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase co-factor, a viral protein chaperone and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.

    IMPORTANCE Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function; yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures for homologous proteins in measles, mumps and Nipah viruses. Distinct oligomeric states of Marburg and Ebola virus VP35s may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of mechanisms governing VP35's functions and inform the design of therapeutics.

  • An amino acid in the stalk domain of N1 neuraminidase is critical for enzymatic activity [PublishAheadOfPrint]

  • Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homo-tetramer with each monomer consisting of a transmembrane region, a stalk and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage; A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2) with almost undetectable NA enzymatic activity compared to the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

    IMPORTANCE Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized, however the importance of specific mutations in the stalk domain are unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

  • One-Health: A Safe, Efficient Dual-use Vaccine for Humans and Animals against MERS-CoV and Rabies Virus. [PublishAheadOfPrint]

  • The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. No treatment options against MERS-CoV exist for humans or animals and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV Spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wildtype MERS-CoV S protein however it showed significantly reduced viral titers compared to the parental RABV strain and only low incorporation of full-length MERS-CoV spike (S) into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to similar titers of the parental vaccine vector BNSP333, and the RABV G/MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, the chemically inactivated BNSP333-S1 induced high titer neutralizing antibodies. Next, we challenged both the vaccinated mice and control mice with MERS-CoV after adenoviral transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor, and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that the vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by significantly lower MERS-CoV titers and MERS-CoV viral and mRNA levels in the challenged mice compared to unvaccinated controls. These data establish that an inactivated RABV/MERS-S-based vaccine may be effective for use in animals and humans in areas with endemic MERS-CoV infections.

    IMPORTANCE Rabies virus-based vectors have proven to be efficient dual vaccines against rabies and emergent infectious diseases like Ebola virus. Here we show that inactivated rabies virus particles containing MERS-CoV spike S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, like camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases.

  • Structure of the opening reading frame 49 protein encoded by Kaposi's sarcoma associated herpesvirus (KSHV) [PublishAheadOfPrint]

  • Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for the herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in KSHV. The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to up-regulate lytic replication in KSHV by enhancing the activities of the replication and transcription activator (RTA). We have solved the crystal structure of ORF49KSHV protein to a resolution of 2.4 AAring;. ORF49KSHV protein has a novel fold consisting of 12 alpha helices bundled into two pseudo-domains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of ORF49KSHV protein in the gamma herpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions suggesting that they are more likely to play important structural roles rather than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. ORF49KSHV protein is stabilized in a dose-responsive manner by double stranded oligonucleotides suggesting actual DNA interaction and binding. Bio-layer interferometry studies also demonstrated that ORF49KSHV protein binds these oligonucleotides.

    Importance Kaposi's sarcoma associated herpesvirus (KSHV) is a tumorigenic gamma herpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle but when it is re-activated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this re-activation but their exact roles are still largely unknown. In this study, we report the protein crystal structure of the open reading frame 49 encoded by KSHV (ORF49KSHV). Possible regions for protein interaction that could harbor functional importance were found on the protein surface of ORF49KSHV. This led to the discovery of novel DNA binding properties by ORF49KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49KSHV were also established with the structure.

  • Clinically approved ion channel inhibitors close gates for hepatitis C virus -and open doors for drug repurposing in viral infectious diseases [PublishAheadOfPrint]

  • Chronic hepatitis C virus (HCV) infection causes severe liver disease and affects ca. 146 million individuals. Novel directly acting antivirals targeting HCV have revolutionized treatment. However, high costs limits access to therapy. Recently, several related drugs used in humans to treat allergies or as neuroleptics emerged as potent HCV cell entry inhibitors. Insights into their antiviral mode of action may access opportunities for drug repurposing in hepatitis C and possibly other important human viral infections.

  • ZAPS inhibits influenza A viral protein expression and is antagonized by the virus encoded NS1 [PublishAheadOfPrint]

  • Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses. There are two ZAP isoforms arising from alternative splicing, which differ only at the C-termini. It was recently reported that the long isoform (ZAPL) promotes proteasomal degradation of influenza A virus (IAV) proteins PA and PB2 through the C-terminal PARP-domain, which is missing in the short form (ZAPS), and that this antiviral activity is antagonized by the viral protein PB1. Here, we report that ZAP inhibits IAV protein expression in a PARP domain-independent manner. Overexpression of ZAPS inhibited the expressions of PA, PB2 and NA and downregulation of the endogenous ZAPS enhanced their expressions. We show that ZAPS inhibited PB2 protein expression by reducing the encoding viral mRNA levels and repressing its translation. However, downregulation of ZAPS only modestly enhanced the early stage of the viral replication. We provide evidence showing that the antiviral activity of ZAPS is antagonized by the viral protein NS1. A recombinant IAV carrying a NS1 mutant that lost the ZAPS-antagonizing activity replicated better in ZAPS-deficient cells. We further provide evidence suggesting that NS1 antagonizes ZAPS by inhibiting its binding to target mRNA. These results uncover a distinct mechanism underlying the interactions between ZAP and IAV.

    IMPORTANCE ZAP is a host antiviral factor that has been extensively reported to inhibit the replication of certain viruses through repressing the translation and promoting the degradation of the viral mRNAs. There are two ZAP isoforms, ZAPL and ZAPS. ZAPL was recently reported to promote IAV protein degradation through the PARP domain. Whether ZAPS, which lacks the PARP domain, inhibits IAV and the underlying mechanisms remained to be determined. Here we show that ZAPS post-transcriptionally inhibits IAV protein expressions. This antiviral activity of ZAP is antagonized by the viral protein NS1. That ZAP uses two distinct mechanisms to inhibit IAV infection and the virus evolved different antagonists suggest an important role of ZAP in the host efforts to control IAV infection and the importance of the threat of ZAP to the virus. The results reported here help to comprehensively understand the interactions between ZAP and IAV.

  • Molecular architecture of the cleavage-dependent mannose patch on a soluble HIV-1 envelope glycoprotein trimer [PublishAheadOfPrint]

  • The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage induces a compact structure, which not only renders the spike capable of fusion but also limits further maturation of its extensive glycosylation. The redirection of the glycosylation pathway to preserve underprocessed oligomannose-type glycans is an important feature in immunogen design as glycans contribute to or influence the epitopes of numerous broadly neutralizing antibodies. Here, we compare quantitative site-specific analysis of a recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a detailed molecular map of trimer-associated glycan remodelling which forms a localised subdomain of the native mannose patch. The formation of native trimers is a critical design feature in shaping the glycan epitopes presented on recombinant vaccine candidates.

    IMPORTANCE The envelope spike of the human immunodeficiency virus (HIV-1) is a target for antibody-based neutralization. In some patients infected with HIV-1, highly potent antibodies have been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 vaccine research to elicit these antibodies by immunization with recombinant mimics of the viral spike. These antibodies have evolved to recognize the dense array of glycans that coat the surface of the viral molecule. We show how the structure of these glycans are shaped by steric constraints imposed upon them by the native folding of the viral spike. This information is important in guiding the development of vaccine candidates.

  • Structure of a Human Astrovirus Capsid - Antibody Complex and Mechanistic Insights into Virus Neutralization [PublishAheadOfPrint]

  • Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immune compromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap with the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCE Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  • Phosphorylation of the bovine papillomavirus E2 protein on tyrosine regulates its transcription and replication functions [PublishAheadOfPrint]

  • Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Post-translational modifications (PTM) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event of a conserved tyrosine (Y) in the bovine papillomavirus type 1 (BPV-1) E2 protein at amino acid 102. While its phospho-deficient mutant phenylalanine (F) activated both transcription and replication in luciferase reporter assays, the mutant that may act as a phospho-mimetic, Y102 to glutamate (E), lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1, however in contrast, Y102E associated with only a subset and was unable to bind to E1. While Y102F fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.

    Author Summary Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understanding the viral replicative cycle nndash; and potential therapies to target it nndash; is examining post-translational modification of viral proteins and its effect on function. Here we have discovered that the viral transcription and replication regulator bovine papillomavirus type 1 (BPV-1) E2 is phosphorylated at tyrosine residue Y102. While a phospho-deficient mutant at this site was fully functional, a phospho-mimetic mutant displayed impaired transcription and replication activity as well as lack of association with certain E2-binding proteins. This study highlights the influence of post-translational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

  • Microminipigs as an animal model for influenza A virus infection. [PublishAheadOfPrint]

  • Pigs are considered a mixing vessel for the generation of novel pandemic influenza A viruses through reassortment because of their susceptibility to both avian and human influenza viruses. However, experiments to understand reassortment in pigs in detail have been limited because experiments with regular-sized pigs are difficult to do. Miniature pigs have been used as an experimental animal model, but, they are still large and require relatively large cages for housing. The microminipig is one of the smallest miniature pigs used for experiments. Introduced in 2010, microminipigs weight around 10 kg at an early stage of maturity (6- to 7-months old), and are easy to handling. To evaluate the microminipig as an animal model for influenza A virus infection, we compared the receptor distribution of ten-week-old male pigs (Yorkshire Large White) and microminipigs. We found that both animals have SAaalpha;2,3Gal and SAaalpha;2,6Gal in their respiratory tract, with similar distribution of both receptor types. We further found that the sensitivity of microminipigs to influenza A viruses was the same as that of larger miniature pigs. Our findings indicate that the microminipig could serve as a novel model animal for influenza A virus infection.

    IMPORTANCE The microminipig is one of the smallest miniature pigs in the world, and is used as an experimental animal model for life science research. In this study, we evaluated the microminipig as a novel animal model for influenza A virus infection. The distribution of influenza virus receptors in the respiratory tract of the microminipig was similar to that of the pig, and the sensitivity of microminipigs to influenza A viruses was the same as that of miniature pigs. Our findings suggest that microminipigs represent a novel animal model for influenza A virus infection.

  • Porcine epidemic diarrhea virus (PEDV) 3C-Like protease-mediated nucleocapsid processing: A possible link to viral cell-culture adaptability [PublishAheadOfPrint]

  • Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in newborn piglets, leading to massive loss to the swine industry worldwide during recent epidemics. Intense research effort is now focusing on defining viral characteristics that confer growth advantage, pathogenicity or cell adaptability in order to better understand PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we reported a unique phenomenon of PEDV nucleocapsid (N) cleavage by PEDV-encoded 3C-like protease (3Cpro) during infection. Identification of the 3Cpro cleavage site at the C-terminus of N supported earlier observations that PEDV 3Cpro showed slightly different substrate requirement from SARS CoV 3Cpro and revealed greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in the emerging field isolates. Remarkably, reverse genetics-derived cell-culture adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells when compared to the wild-type virus. These observations altogether shed new light into investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation.

    IMPORTANCE Recurrent PEDV outbreaks have resulted in enormous economic losses to the swine industries worldwide. To gain the upper hand in combating the disease, it is necessary to understand how the virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses use to survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsid of some PEDV strains is proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests replication advantage provided by the cleavage event at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

  • TIM-1 promotes hepatitis C virus cell attachment and infection [PublishAheadOfPrint]

  • Human TIM and TAM family proteins were recently found to serve as phosphatidylserine receptors which promote infection of many different viruses including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection of hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and the subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be completely restored by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by phosphatidylserine (PS) but not phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS-dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS ligand exposed on the HCV envelope.

    IMPORTANCE TIM family proteins were recently found to enhance infection of many different viruses including several members of the Flaviviridae family. However, their importance in HCV infection has not been experimentally examined. The TIM family proteins include three members in humans, TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1 but not TIM-3 or TIM-4 promotes HCV infection by functioning as an HCV attachment factor. The knockout of TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand PS may serve as novel targets for antiviral intervention.

  • Evidence that Hsc70 is associated with CNV particles and plays a role in CNV particle disassembly [PublishAheadOfPrint]

  • Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a non-enveloped (+)ssRNA virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog, Hsc70-2, copurifies with CNV particles. Virus overlay and immunogold labelling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus indicating an intimate association with particles. To investigate a possible role of Hsc70-2 in particle disassembly, we show that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions compared to prebleed control antibody on Chenopodium quinoa. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant N. benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus.

    IMPORTANCE Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of non-enveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2, is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor, Hsc70-2, in CNV disassembly.

  • Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses [PublishAheadOfPrint]

  • Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the US in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189 and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189 and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses.

    IMPORTANCE Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the US since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the US swine population in the mid-1990s, but are different from both these 1990s ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.

  • Evolution and cryo-EM capsid structure of a North American bat adenovirus and its relationship to other mastadenoviruses [PublishAheadOfPrint]

  • Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV 250-A) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. BtAdV 250-A exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy (cryo-EM) reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV.

    IMPORTANCE Although many adenoviruses are host-specific and likely co-diverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time-scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.

  • Virion background and efficiency of virion incorporation determine susceptibility of SIV-Env-driven viral entry to inhibition by IFITM proteins [PublishAheadOfPrint]

  • Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate-specific, raising the question which parameters impact IFITM-sensitivity. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact IFITM-sensitivity of viral entry.

    IMPORTANCE The host cell encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact IFITM sensitivity of viral entry. These results show for the first time that determinants other than the viral envelope protein can impact IFITM sensitivity and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins.

  • Restarting Lytic Gene Transcription at the Onset of Herpes Simplex Virus Reactivation. [PublishAheadOfPrint]

  • Herpes simplex virus type (HSV) establishes a latent reservoir in neurons of human peripheral nerves. In this quiescent state the viral genome persists as a circular, histone-associated episome and transcription of viral lytic-cycle genes is largely suppressed through epigenetic processes. Periodically latent virus undergoes reactivation whereby lytic genes are activated and viral replication occurs. In this GEM we review recent evidence that mechanisms governing the initial transcription of lytic genes are distinct from those of de novo infection and directly link reactivation to neuronal stress response pathways. We also discuss evidence that lytic cycle gene expression can be uncoupled from the full reactivation program, arguing for a less sharply bimodal definition of latency.

  • Influenza overcomes cellular blocks to productively replicate impacting macrophage function [PublishAheadOfPrint]

  • Whether influenza replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks; the first block is during viral entry where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion resulting in lysomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG opsonized bioparticles and Fc receptors CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and may play an important role in pathogenesis during infection in vivo.

    IMPORTANCE Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet to be identified mechanism(s) to overcome a post-nuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.

  • Heterologous immunity and persistent murine cytomegalovirus infection [PublishAheadOfPrint]

  • One's history of infections can affect the immune response to unrelated pathogens and influence disease outcome through the process of heterologous immunity. This can occur after acute viral infections, such as lymphocytic choriomeningitis virus (LCMV) and vaccinia virus, where the pathogens are cleared, but it becomes a more complex issue in the context of persistent infections. In this study, murine cytomegalovirus (MCMV) was used as a persistent infection model to study heterologous immunity with LCMV. If mice were previously immune to LCMV and then infected with MCMV (LCMV+MCMV), they had more severe immunopathology, enhanced viral burden in multiple organs, and suppression of MCMV-specific T cell memory inflation. MCMV infection initially reduced the numbers of LCMV-specific memory T cells, but continued MCMV persistence did not further erode memory T cells specific to LCMV. When MCMV infection was given first (MCMV+LCMV), the magnitude of the acute T cell response to LCMV declined with age, though this age-dependent decline was not dependent on MCMV. However, some of these MCMV persistently infected mice with acute LCMV infection (7 of 36) developed a robust immunodominant CD8 T cell response apparently cross-reactive between a newly defined putative MCMV epitope sequence M57727-734 and the normally subdominant LCMV epitope L2062-2069, indicating a profound private specificity effect in heterologous immunity between these two viruses. These results further illustrate how a history of an acute or a persistent virus infection can substantially influence the immune responses and immune pathology associated with acute or persistent infections with an unrelated virus.

    IMPORTANCE This study extends our understanding of heterologous immunity in the context of persistent viral infection. The phenomenon has been studied mostly with viruses such as LCMV that are cleared but the situation can be more complex with a persistent virus such as MCMV. We found that the history of LCMV infection intensifies MCMV immunopathology, enhances MCMV burden in multiple organs, and suppresses MCMV-specific T cell memory inflation. In the reverse infection sequence, we show that some of the long-term MCMV-immune mice mount a robust CD8 T cell cross-reactive response between a newly defined putative MCMV epitope sequence and a normally subdominant LCMV epitope. These results further illustrate how a history of infection can substantially influence the immune responses and immune pathology associated with infections with an unrelated virus.

  • Subcellular localizations of RIG-I, TRIM25 and MAVS complexes [PublishAheadOfPrint]

  • The RIG-I signaling pathway is essential for the recognition of viruses and the initiation of host IFN-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds MAVS, leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway, through the use of Bimolecular Fluorescence Complementation (BiFC) and super resolution microscopy. Dimers of RIG-I, TRIM25 and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25 mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus (HCV) redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. By contrast, the influenza A virus (IAV) NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm, and inhibits the formation of TRIM25 homocomplexes, but not of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25 and MAVS, in the presence and absence of two viral IFN antagonistic proteins.

    IMPORTANCE The first line of defense against viral infections is the innate immune response. Viruses are recognized by Pathogen Recognition Receptors (PRR), like the RIG-I like receptor (RLR) family, that activate a signaling cascade that induces IFN production. In the present study we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal leading to an efficient antiviral state. In addition, this work presents a new means by how, when and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.

  • Replicating Single-cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses [PublishAheadOfPrint]

  • Head to head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrate these viral vectors can mediate more potent protection against influenza infection in animal models. In most cases, Ad vaccines are engineered to be replication defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent, but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication, but avoid production of infectious virions, we developed "single cycle" adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and Rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin gene from influenza A/PR/8/34. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad vaccinated cotton rats had markedly lower influenza titers than RD-Ad vaccinated animals after challenge with influenza A/PR/8/34. These data suggest that SC-Ads may be more potent vaccine platforms that conventional RD-Ad vectors and may have utility as "needle-free" mucosal vaccines.

    Importance Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ad). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ad generate markedly more influenza hemagglutin protein and require substantially less vector to generate the same immune responses as RD-Ad vector. SC-Ad therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.

  • Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells [PublishAheadOfPrint]

  • Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV), but not Moloney murine leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells.

    Importance Human embryonic carcinoma (EC) cells are shown to restrict the expression of murine leukemia virus genomes but not retroviral genomes of the lentiviral or betaretroviral families. The block occurs at the level of transcription, and is accompanied by the deposition of repressive histone marks and methylation of the integrated proviral DNA. The host machinery required for silencing in human EC cells is distinct from that in murine EC cell lines: the histone methyltransferase SETDB1 is required, but the widely-utilized corepressor TRIM28/Kap1 is not. A transcriptional enhancer element from the Mason-Pfizer monkey virus can override the silencing and promote transcription of chimeric proviral DNAs. The findings reveal novel features of human EC gene regulation not present in their murine counterparts.

  • The dual roles of the HA segment-specific noncoding nucleotides in the extended duplex region of the influenza A virus RNA promoter [PublishAheadOfPrint]

  • We recently reported that the segment-specific noncoding regions (NCRs) of the HA and NA segments are subtype-specific, varying significantly in sequence and length at both the 3rrsquo; and 5rrsquo; ends. Interestingly, we found that nucleotides "CC" at positions 13-14 at the 3rrsquo; end and "GUG" at positions 14rrsquo; -16rrsquo; at the 5rrsquo; end are absolutely conserved among all HA subtype-specific NCRs. These HA segment-specific NCR nucleotides are located in the extended duplex region of the viral RNA promoter. In order to understand the significance of these highly conserved HA segment-specific NCR nucleotides in the virus life cycle, we performed extensive mutagenesis on the HA segment-specific NCR nucleotides and studied their functional significance in regulating influenza A virus replication in the context of the HA segment with both RNP reconstitution and virus infection systems. We found that the base-pairing at 3rrsquo;13-5rrsquo;14rrsquo; positions is critical for RNA promoter activity while the identity of the base pair is critical in determining HA segment packaging. Moreover, the identity of the residue at 3rrsquo;14 is more functionally important in regulating virus genome packaging than in regulating viral RNA synthesis. Taken together, these results demonstrated that the HA segment-specific NCR nucleotides in the extended duplex region of the promoter not only form part of the promoter, but also play a key role in controlling virus selective genome packaging.

    IMPORTANCE The segment-specific complementary nucleotides (13-15 in the 3rrsquo; end and 14rrsquo; -16rrsquo; in the 5rrsquo; end) in the extended duplex region of the influenza virus RNA promoter vary significantly among different segments and have rarely been studied. We here performed mutagenesis analysis of the highly conserved HA segment-specific nucleotides in the extended duplex region and examined their effects on virus replication in the context of the WSN virus infection. We found that these HA segment-specific nucleotides, not only act as a part of the RNA promoter, but also play a critical role in HA segment packaging. Therefore, we showed experimentally, for the first time, the requirement of the nucleotides in the extended duplex region for the RNA promoter, but also identified specific noncoding residues in regulating HA segment packaging. This work has implications for the development of attenuated vaccine strains and for elucidation the mechanisms of the virus genome packaging.

  • Replication-deficient particles: New insights into the next generation of bluetongue virus vaccines [PublishAheadOfPrint]

  • Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe haemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (DISC) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature and desiccation were determined and their protective efficacies in animals confirmed. Further, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge or after five months' challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than sheep. Our data support and validate the suitability of these virus strains as the next generation vaccines for BTV.

    Importance Bluetongue (BT) is a debilitating and in many cases a lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidences and support the suitability of these virus strains as the next generation BTV vaccines.

  • A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure [PublishAheadOfPrint]

  • Porcine circovirus associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus type 2 (PCV2) is an essential component of PCVAD although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 share only 37% and 55% identity to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57x107 genomic copies/ml) and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) on sow tissues identified PCV3 antigen in skin, kidney, lung and lymph nodes localized in typical PDNS lesions including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis and bronchinterstitial pneumonia. Further study of archived PDNS tissues, that were negative for PCV2 by IHC, identified 45 of 48 were PCV3 positive by qPCR with 60% of a subset also testing positive for PCV3 by IHC. Analysis by qPCR of 271 porcine respiratory disease diagnostic submissions identified 34 PCV3 positive cases (12.5%), and ELISA detection of anti-PCV3 capsid antibodies in sera found 46 positive samples of 83 tested (55%). These results suggest PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Due to the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD.

    IMPORTANCE While first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990's, an epidemic of severe systemic disease due to porcine circovirus type 2 (PCV2) spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3), which is distantly related to known circoviruses, was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid was observed in aborted fetuses by quantitative PCR (qPCR) and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC). PCV3 was also identified in archival PDNS diagnostic cases which previously tested negative for PCV2 using IHC. The emergence of PCV3 warrants further investigation.

  • {varphi}X174 procapsid assembly: the effects of an inhibitory external scaffolding protein and resistant coat proteins in vitro [PublishAheadOfPrint]

  • During X174 morphogenesis, 240 copies of the external scaffolding protein D organize twelve pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation arose that greatly increased strain fitness. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context.

    IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an anti-viral protein, and then evolve a productive use for that protein. Moreover, our results indicate that respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two scaffolding protein system. In one-scaffolding protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ooslash;X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

  • CD8+ T cells and Macrophages Regulate Pathogenesis in a Mouse Model of MERS-CoV Disease [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) is an important emerging pathogen that was first described in 2012. While the cell surface receptor for MERS-CoV has been identified as dipeptidyl peptidase IV (DPP4), the mouse DPP4 homologue does not allow for virus entry into cells. Therefore, development of mouse models of MERS-CoV has been hampered by the fact that MERS-CoV does not replicate in commonly available mouse strains. We have previously described a mouse model in which mDPP4 was replaced with hDPP4 such that hDPP4 is expressed under the endogenous mDPP4 promoter. In this study, we used this mouse model to analyze the host response to MERS-CoV infection using immunological assays and transcriptome analysis. Depletion of CD4+T cells, CD8+ T cells or macrophages has no effect on MERS-CoV replication in the lungs of infected mice. However, we found that depletion of CD8+ T cells protects and depletion of macrophages exacerbates MERS-CoV induced pathology and clinical symptoms of disease. Overall, we demonstrate an important role for the inflammatory response in regulating MERS-CoV pathogenesis in vivo.

    Importance The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a highly pathogenic respiratory virus that emerged from zoonotic sources in 2012. Human infections are still occurring throughout Saudi Arabia at a 38% case fatality rate, with the potential for worldwide spread via air travel. In this work, we identify the host response to the virus and identify inflammatory pathways and cell populations that are critical for protection from severe lung disease. By understanding the immune response to MERS-CoV we can develop targeted therapies to inhibit pathogenesis in the future.

  • Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge. [PublishAheadOfPrint]

  • African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in highly virulent ASFVs Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here we report the construction of a double gene deletion recombinant virus, ASFV-G-9GL/UK. When administered intramuscularly (IM) to swine there is no induction of disease even at high doses (106 HAD50). Importantly, animals infected with 104 HAD50 of ASFV-G-9GL/UK were protected as early as 14 days post-inoculation when challenged with ASFV-G. Presence of protection correlates with appearance of serum anti-ASFV antibodies but not with virus specific circulating ASFV-specific INF- producing cells. ASFV-G-9GL/UK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as soon as 2 weeks post-vaccination.

    Importance: Currently there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating to the swine industry, and are caused by circulating strains of African swine fever virus. Here we report about a putative vaccine derived from a current circulating strain but containing two deletions in two separate areas of the virus, allowing for increased safety. Using this genetically modified virus, we are able to vaccine and protect swine from developing ASF. We were able to achieve protection from disease as early as two weeks after vaccination, even when pigs were exposed to a higher than normal concentration of ASFV.

  • The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella Zoster Virus Cell-Cell Fusion Regulation and Infection [PublishAheadOfPrint]

  • The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytia formation, a characteristic of Varicella Zoster Virus (VZV) pathology in skin and sensory ganglion. VZV syncytia formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation as alanine substitutions induced a 440% increase in fusion compared to the wildtype gBcyt while arginine substitutions had wildtype-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytia formation, reduced VZV titers (-1.5log10) and smaller plaques compared to pOka. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5log10 reduction in titers and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques, but differed in syncytia morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt.

    IMPORTANCE Varicella Zoster Virus (VZV) is a ubiquitous pathogen that causes chicken pox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN) that has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed as the current vaccine is not recommended for immune compromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue which has been proposed to be a contributing factor to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.

  • Dysregulated Glycoprotein B Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription During Infection [PublishAheadOfPrint]

  • The highly conserved herpesvirus glycoprotein complex, gB/gH-gL, mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multi-nucleated cells, or syncytia, during the infection of human tissues but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. The gBcyt regulation is necessary for VZV pathogenesis as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt regulated fusion was investigated by comparing melanoma cells infected with wild type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytia formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization and rapid displacement of nuclei to dense central structures when compared to pOka using live cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using RNA-seq to identify viral and cellular responses induced when the gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and glycoproteins, gC, gE and gI, was significantly reduced at 36 hours post infection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate the gBcyt in the regulation gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection.

    IMPORTANCE The highly infectious, human restricted pathogen, varicella-zoster virus (VZV), causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigates the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live cell imaging and next generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

  • IRF-1 and type I interferon cooperate to control acute gammaherpesvirus infection. [PublishAheadOfPrint]

  • Gammaherpesviruses are ubiquitous pathogens that establish life-long infection in ggt;95% of adults worldwide, and are associated with a variety of malignancies. Coevolution of gammaherpesviruses with their hosts has resulted in an intricate relationship between the virus and the host immune system, and perturbation of the virus-host balance results in pathology. Interferon regulatory factor 1 (IRF-1) is a tumor suppressor that is also involved in the regulation of innate and adaptive immune responses. Here we show that type I Interferon (IFN) and IRF-1 cooperate to control acute gammaherpesvirus infection. Specifically, we demonstrate that a combination of IRF-1 and type I IFN signaling ensures host survival during acute gammaherpesvirus infection and supports IFN gamma-mediated suppression of viral replication. Thus, our studies reveal an intriguing crosstalk between IRF-1 and type I and II IFN in the induction of the antiviral state during acute gammaherpesvirus infection.

    IMPORTANCE Gammaherpesviruses establish chronic infection in a majority of adults and this long-term infection is associated with virus-driven development of a range of malignancies. In contrast, brief period of active gammaherpesvirus replication during acute infection of a naïve host is subclinical in most individuals. Here we discovered that a combination of type I interferon (IFN) signaling and Interferon Regulatory Factor-1 (IRF-1) expression is required to ensure survival of gammaherpesvirus-infected host past the first 8 days of infection. Specifically, both type I IFN receptor and IRF-1 expression potentiated antiviral effects of type II IFN to restrict gammaherpesvirus replication in vivo, in the lungs, and in vitro, in primary macrophage cultures.

  • Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway [PublishAheadOfPrint]

  • Hypoxia inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target in viral manipulation of the cellular environment. Under normoxic conditions HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, Factor Inhibiting HIF (FIH) has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis we identified the five ANK proteins of the parapoxvirus, orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, co-immunoprecipitation of FIH with each of the ORFV ANK proteins was detected and with one representative ORFV ANK protein the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed co-localization of FIH and the viral ANK proteins. In addition mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro. While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in de-repression of HIF-1aalpha; activity in reporter gene assays. Furthermore, ORFV-infected cells showed up-regulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to de-repression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family.

    IMPORTANCE The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus, orf virus (ORFV) is able to upregulate Hypoxia Inducible Factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator, Factor Inhibiting HIF (FIH). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism and reveal a new role for the ANK family of poxvirus proteins.

  • Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) to Facilitate the IN Assembly and Non-enzymatic Function Through the IN Precursor Form with Reverse Transcriptase [PublishAheadOfPrint]

  • Non-enzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the non-enzymatic roles of IN, we performed genetic analyses of HIV-1 IN focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Substitution of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, substitution of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. Rescue experiment of RT-IN-deleted (RTIN) HIV-1 by supplying IN and RT in trans revealed that the non-enzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form.

    IMPORTANCE Inhibitors of the IN enzymatic strand-transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINs affect the non-enzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its non-enzymatic function. Our results provide a novel mechanistic insight into the non-enzymatic function of HIV-1 IN and its prevention.

  • JunD/AP-1 antagonizes the induction of DAPK1 to promote the survival of v-Src transformed cells [PublishAheadOfPrint]

  • The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant-negative mutant TAM67 induced senescence, adipogenesis or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEF) whereas inhibition of JunD by shRNA specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEF expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe-sets up-regulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe-sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPbbeta;-regulated mediator of apoptosis in IFN-induced cell death. Here we show that inhibition of DAPK1 abrogates cell-death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPbbeta; was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPbbeta; abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by ChIP assays, JunD was not detected on the DAPK1 promoter in any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells.

    IMPORTANCE Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-B and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumour suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumour suppressor function of DAPK1.

  • Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the cGMP/PKG and NF-{kappa}B Signaling Pathway [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research had demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV, however, it did not affect PRRSV entry of host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cGMP/PKG signaling pathway. CO significantly inhibits PRRSV-induced NF-B activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced pro-inflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication, but also suggest potential new control measures for future PRRSV outbreaks.

    IMPORTANCE PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication, but also suggest potential new control measures for future PRRSV outbreaks.

  • The cellular DNA helicase ChlR1 regulates chromatin and nuclear matrix attachment of the human papillomavirus type 16 E2 protein and high copy viral genome establishment [PublishAheadOfPrint]

  • In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes to daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus type 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here, we show that the HPV16 E2 mutation Y131A has significantly reduced binding to ChlR1, but retains transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A shows a marked change in the localization of the protein. In comparison to wild type E2, the chromatin-bound pool of E2Y131A is decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicates that the shift in subcellular localization of E2Y131A occurs in mid-S phase. A similar alteration between the subcellular pools of E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than wild type HPV16 genomes, and were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes.

    IMPORTANCE Infection with high-risk human papillomavirus (HPV) types is a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA HPV genome persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by simultaneously binding to the viral genome and chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for the loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here, we have identified a mutation in HPV16 E2 that abrogates interaction with ChlR1 and show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

  • A Helical Short-Peptide Fusion Inhibitor with Highly Potent Activities against HIV-1, HIV-2 and Simian Immunodeficiency Virus [PublishAheadOfPrint]

  • HIV-2 has already spread to different regions worldwide and currently about 1-2 million people have been infected, calling for new antiviral agents that are effective on both HIV-1 and HIV-2 isolates. T-20 (Enfuvirtide), a 36-mer peptide derived from the C-terminal heptad repeat region (CHR) of gp41, is the only clinically approved HIV-1 fusion inhibitor, but it easily induces drug-resistance and is not active on HIV-2. In this study, we firstly demonstrated that the M-T hook structure was also a vital strategy to enhance the binding stability and inhibitory activity of diverse CHR-based peptide inhibitors. Then, we designed a novel short-peptide (23-mer) termed 2P23 by introducing the M-T hook structure, HIV-2 sequences and llsquo;salt-bridges'-forming residues. Promisingly, 2P23 was a highly stable helical peptide with high binding to the surrogate targets derived from HIV-1, HIV-2 and simian immunodeficiency virus (SIV). In consistence, 2P23 exhibited potent activity in inhibiting diverse subtypes of HIV-1 isolates, T-20-resistant HIV-1 mutants, and a panel of primary HIV-2 isolates, HIV-2 mutants and SIV isolates. Therefore, we conclude that 2P23 has high potential to be further developed for clinical use and it is also an ideal tool for exploring the mechanisms of HIV-1/2 and SIV-mediated membrane fusion.

    IMPORTANCE The peptide drug T-20 is the only approved HIV-1 fusion inhibitor, but it is not active on HIV-2 isolates which have currently infected 1-2 million people and continuingly spread worldwide. Recent studies have demonstrated that the M-T hook structure can greatly enhance the binding and antiviral activities of gp41 CHR-derived inhibitors, especially for short-peptides otherwise inactive. By combining the hook structure, HIV-2 sequence and llsquo;salt-bridge'-based strategies, the short-peptide 2P23 has been successfully designed. 2P23 exhibits prominent advantages over many other peptide fusion inhibitors, including its potent and broad activity on HIV-1, HIV-2 and even SIV isolates, its stability as a helical, oligomeric peptide, and its high binding to diverse targets. The small size of 2P23 would benefit its synthesis and significantly reduce production cost. Therefore, 2P23 is an ideal candidate for further development and it also provides a novel tool for studying HIV-1/2 and SIV-mediated cell fusion.

  • Naf1 regulates HIV-1 latency by suppressing viral promoter-driven gene expression in primary CD4+ T cells [PublishAheadOfPrint]

  • HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating the LTR activity contribute to HIV-1 latency and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleo-cytoplasmic shuttling protein and regulates multiple cellular signal pathways and HIV-1 production. We have recently reported that the nucleus-located Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to the accumulated Gag production. Here, we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both HIV-1 latently infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo. Our findings provide new insights into a better understanding of HIV-1 latency, and implicate that the inhibition of Naf1 activity to activate HIV-1 latently infected cells may be a potential therapeutic strategy.

    IMPORTANCE HIV-1 latency is mainly characterized by a reversible silencing of long terminal repeats (LTR) promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that host protein Naf1 inhibited HIV-1-LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into a better understanding of host-modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting Naf1 protein.

  • CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo [PublishAheadOfPrint]

  • Human infections with influenza viruses exhibit mild to severe clinical outcome as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin member 5A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of pro-inflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced TNF-aalpha; and IP-10 but elevated IFN-aalpha; compared to wild-type mice. The heightened type-I IFN response in the macrophages of CLEC5A-deficient mice was associated with up-regulated TLR3 mRNA after treatment with double stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of pro-inflammatory cytokines, decreased immune cell infiltration in the lungs and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered as a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections.

    IMPORTANCE Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response at gate to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of pro-inflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival than the wild-type mice, despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells may reduce immunopathogenesis after influenza infections.

  • Efficient transformation of primary human mesenchymal stromal cells by adenovirus early region 1 oncogenes [PublishAheadOfPrint]

  • Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible for transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSC), can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficient as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transform cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture at a high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSC will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well.

    IMPORTANCE It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type, which is susceptible for HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type, is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSC) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression, that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic or osteogenic lineage. Hence, primary hMSC represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

  • Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone [PublishAheadOfPrint]

  • Zika virus (ZIKV; Family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated comparable infection and transmission rates in Aedes aegypti mosquitoes compared to the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis.

    IMPORTANCE ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barreeacute; syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with similar in vitro and in vivo characteristics to the primary isolate, providing a critical tool to study ZIKV infection and disease.

  • Broadly Neutralising Antibodies Display Superior Potential Over Non-Neutralising Antibodies in Preventing HIV-1 infection of Mucosal Tissue [PublishAheadOfPrint]

  • Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralisation is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Non-neutralising antibodies (nnAbs) may also impact on mucosal transmission events through Fc-gamma receptor (FcR)-mediated inhibition. Here, a panel of broadly neutralising (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for their ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralisation potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4bs-specific bnAbs, and in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. MPER (2F5) and outer-domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) were more variable. In contrast, nnAb alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest tissue resident effector cell numbers and low FcR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over non-neutralising antibodies for preventing HIV-1 infection at the mucosal portals of infection.

    IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting protection might be mediated in the absence of classical neutralisation. To aid vaccine design and selection of antibodies for use in passive protection strategies we assessed a range of bnAbs and nnAbs for their potential to block ex-vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralising antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralising antibodies.

  • Host cellular protein TRAPPC6A{Delta} interacts with influenza A virus M2 protein and regulates viral propagation by modulating M2 trafficking [PublishAheadOfPrint]

  • Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 post-translational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal deletion isoform TRAPPC6A delta (TRAPPC6A) interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6A in the viral life cycle was investigated by knockdown of endogenous TRAPPC6A with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6A. The results indicated that TRAPPC6A, through its interaction with M2, slows down M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice.

    IMPORTANCE Influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the post-translational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal deletion isoform TRAPPC6A as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factors, TRAPPC6A/TRAPPC6A. Importantly, we discovered that TRAPPC6A can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

  • Differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus, EqHV). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5-98.2% identical in their translated polyprotein sequences, making virus/host co-speciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV.

    IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus, EqHV). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys and EqHV may have co-speciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV co-speciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

  • The long and complicated relationship between Epstein-Barr virus and epithelial cells [PublishAheadOfPrint]

  • The role of epithelial cells in infection and persistence of Epstein-Barr virus (EBV) has long been difficult to resolve. Recent developments have, however, reinforced both the conclusion that they are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure both survival persistence and spread.

  • Recombinant receptor-binding domains of multiple MERS-coronaviruses induce cross-neutralizing antibodies against divergent human and camel MERS-coronaviruses and antibody-escape mutants [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody-escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014 and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing mAbs and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 mAb-escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains.

    IMPORTANCE MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and camel-RBD, respectively, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody-escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

  • Potent Inhibition of HCMV by Modulating the Cellular SNARE Syntaxin 5 [PublishAheadOfPrint]

  • Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. Although a recent screen identified three viral proteins essential for cVAC formation, less is known about the contribution of cellular factors. We show that HCMV infection increases the protein level of a cellular trafficking factor, Syntaxin 5 (STX5), a member of the syntaxin family of SNARE proteins. STX5 is recruited to the cVAC in infected cells and is required for the efficient production of infectious virions. We find that STX5 is important for normal cVAC morphology and the proper localization of viral proteins. A previously identified inhibitor of trafficking, Retro94, causes the mislocalization of STX5, an altered cVAC morphology and dispersal of viral proteins. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs. We show that this inhibition is conserved among different strains of HCMV, the various cell types that support infection, as well as for murine CMV. Thus, our data identify a key cellular trafficking factor important for supporting HCMV infection.

    IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe disease and mortality in immunocompromised individuals, including organ transplant and AIDS patients. In addition, infection of a developing fetus may result in lifelong complications such as deafness and learning disabilities. Understanding in detail the processes involved in HCMV replication is important for developing novel treatments. One of these essential processes, assembly of infectious virions, takes places in the cytoplasmic viral assembly compartment. We identify a cellular protein, syntaxin 5, important for generating this compartment and show that it is required for the efficient production of infectious virions. We also show that a small molecule that disrupts this protein also significantly reduces the amount of infectious virions that are generated. Thus, by pinpointing a cellular protein that is important in the replication cycle of HCMV, we identified a novel target that can be pursued for therapeutic intervention.

  • Native folding of a recombinant gpE1/gpE2 heterodimer vaccine antigen from a precursor protein fused with Fc IgG [PublishAheadOfPrint]

  • A recombinant HCV-1 (1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high affinity and scalable method for isolating E1E2. We generated an Fc-tag-derived (Fc-d) E1E2 that was selectively captured by Protein G Sepharose with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to GNA purified wild type (WT) E1E2 and exhibited near identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26 and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited similar anti-E2 antibody titers and neutralization of HCV pseudotype viruses to WT E1E2. Competition ELISAs showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d immunized mice exhibited stronger competition for AR3B and AR5A than WT, whereas competition for HC84.26 and AR4A was similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using Protein A/G-based chromatography.

    IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain tagged E1E2 antigen that forms functional heterodimers similar to the native E1E2. Affinity purification and tag removal of the Fc-tagged E1E2 resulted in an antigen with a near identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate our method will provide a means for large scale production of this HCV vaccine candidate.

  • Interleukin-17A Promotes CD8+ T Cell Cytotoxicity to Facilitate West Nile Virus Clearance [PublishAheadOfPrint]

  • CD8+ T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic mediator gene expression and promoting CD8+ T cell cytotoxicity against WNV infection in mice. We find that IL-17A deficient mice (Il17a-/-) are more susceptible to WNV infection and develop a higher viral burden compared to wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo. Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 post-infection, significantly reduces viral burden and increases survival, suggesting a therapeutic potential of IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8+ T cell cytotoxicity, which may have broad implications in other microbial infections and cancers.

    IMPORTANCE Interleukin-17A (IL-17A) and CD8+ T cells regulate diverse immune functions during microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while the CD8+ T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8+ T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection yet remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8+ T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8+ T cell axis may also have broad implications in immunity to other microbial infections and cancers, where CD8+ T cell functions are crucial.

  • Pivotal role of RIP1 and MLKL in neuronal cell death induced by the human neuroinvasive coronavirus OC43 [PublishAheadOfPrint]

  • Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic and neurovirulent properties, highlighting the importance to study the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determine if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process reflecting the severity of neuropathogenesis.

    IMPORTANCE OF THE STUDY Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein is not playing a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (a RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and may induce the production of pro-inflammatory cytokines, it may represent a pro-inflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.

  • An in vitro RNA synthesis assay for rabies virus defines critical ribonucleoprotein interactions for polymerase activity. [PublishAheadOfPrint]

  • We report an in vitro RNA synthesis assay for the RNA dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19-nt of the rabies virus genome we demonstrate that L alone initiates synthesis on naked RNA, and that P serves to enhance initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11-50 of P, and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with that of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P in which the oligomerization domain (OD) and the CTD were replaced by that of VSV P stimulated RABV RdRP activity on naked RNA, but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis extending our knowledge of ribonucleoprotein interactions that are critical for gene expression.

    IMPORTANCE Current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P information on both the structure and function of the L protein are lacking. This study reports the expression and purification of the full-length L protein of RABV and a characterization of its RdRP activity in vitro. The study provides a new assay that has utility for screening inhibitors and understanding their mechanism of action, as well as defining new interactions that are required for RdRP activity.

  • Vietnamese influenza A viruses of swine between 2010 and 2015: multiple introductions of A(H1N1)pdm09 viruses into the pig population and diversifying genetic constellations of enzootic IAV-S [PublishAheadOfPrint]

  • Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North America and pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09-origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as co-circulation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam.

    IMPORTANCE This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S endemic since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.

  • The Potyviral Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways [PublishAheadOfPrint]

  • RNA silencing is an innate antiviral immunity of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, the helper component-proteinase (HC-Pro) and the viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in RNA silencing suppression is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes the reduced level of SUPPRESSOR OF GENE SILENCING3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived siRNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We further present evidence that expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner, RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 is via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in silencing suppression by VPg.

    IMPORTANCE Potyviruses represent the largest group of known plant viruses and cause significant losses in many agriculturally important crops in the world. In order to establish their infections, potyviruses must overcome host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

  • Creating an artificial tail-anchor as a novel strategy to enhance the potency of peptide-based HIV fusion inhibitors [PublishAheadOfPrint]

  • T20 (enfuvirtide) and other peptides derived from the human immunodeficiency virus type 1 (HIV-1) gp41 C-terminal heptad repeat (CHR) region inhibit HIV fusion by binding to the hydrophobic grooves on the N-terminal heptad repeat (NHR)-trimer and blocking the six-helix bundle (6-HB) formation. Several strategies focusing on the binding grooves of NHR-trimer have been adopted to increase antiviral activity of the CHR-peptides. Here we developed a novel and simple strategy to greatly enhance the potency of the existing peptide-based HIV fusion inhibitors. Firstly, we identified a shallow pocket adjacent to the groove in the N-terminal region of NHR-trimer as a new drug target, and then designed several short artificial peptides to fit this target. After adding IDL (Ile-Asp-Leu) to the C-terminus of CHR-peptide WQ or MT-WQ, the conjugated peptide WQ-IDL or MT-WQ-IDL showed much more potent activities than WQ and T20, respectively, in inhibiting HIV-1 IIIB infection. WQ-IDL and MT-WQ-IDL were also more effective than WQ in blocking HIV-1 Env-mediated membrane fusion and had higher binding affinity with NHR-peptide N46. We solved the crystal structure of 6-HB formed by MT-WQ-IDL and N46, and found that besides the N-terminal MT hook-tail, the IDL tail-anchor of MT-WQ-IDL also bind with the shallow hydrophobic pocket outside the groove of the NHR-trimer, resulting in the enhanced inhibition of HIV-1 fusion with the target cell. It is expected that this novel approach can be widely used to improve the potency of the peptidic fusion inhibitors against other enveloped viruses with class I fusion protein.

    IMPORTANCE The hydrophobic groove of human immunodeficiency virus type 1 (HIV-1) gp41 NHR-trimer has been known as the classic drug target to develop fusion inhibitors derived from gp41 CHR. Here we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR-trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C-terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced by 77-fold, much more potent than T20 (enfuvirtide), suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR-peptide fusion inhibitors. This new approach by identifying potential binding pocket outside the traditional target and creating an artificial tail-anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as MERS-CoV.

  • Identification of novel KSHV Orf50 transcripts: discovery of new RTA isoforms with variable transactivation potential [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma, multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes allowing for it to control the switch between latency and virus replication. Here we report the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial cells), 3T12 (mouse fibroblast), and Raw 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 -- 10 residues at the amino-terminus of the protein. Here we show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.

    IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcom-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma, multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication, ane is conserved among all known gammaherpesviruses. Here we show for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of serveral isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression leading to the efficient production of mature virions.

  • Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160 [PublishAheadOfPrint]

  • The envelope (Env) glycoprotein of HIV is expressed on the surface of productively-infected cells, and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties including toxins, drugs, or radionuclides are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g. MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We have evaluated toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in SHIV-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated, initially efficacious, but ultimately limited by immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo, but also highlight potential problems to be addressed.

    IMPORTANCE It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate, consisting of an anti-HIV antibody bound to a toxic moiety, could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab, and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells. But their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating ITs with polyethylene glycol minimizes immunogenicity, as has been demonstrated with other protein therapies.

  • Importance of highly conserved peptide sites of HCMV gO for the formation of the gH/gL/gO complex [PublishAheadOfPrint]

  • The glycoprotein O (gO) is betaherpesvirus-specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids (aa) that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites - containing all 13 highly conserved amino acids - were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of aa181-186 or aa193-198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa108 or aa249-254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was particular as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent, but aa181-198 and cysteines 343, 218 and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is neither required for its incorporation into virions nor for proper function in cell-free infection.

    Importance Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role for the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked, as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL, however non-covalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity.

  • Deep-sequence identification and role in virus replication of a JC virus quasispecies in patients with progressive multifocal leukoencephalopathy [PublishAheadOfPrint]

  • JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nt 3495 in JCV Mad1 resulting in a V to G amino acid substitution at aa position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3%nndash;19% variant frequencies. Transfection of JCV Mad1 DNA having the V392G substitution in TAg into IMR-32 and HEK293 cells resulted in a dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Co-transfection with equal amounts of wild type and mutated JCV genome did not reduce the expression of viral proteins nor viral replication, suggesting that the mutation did not have any dominant negative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited.

    IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has been rarely reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

  • Structural biology of the arterivirus nsp11 endoribonucleases [PublishAheadOfPrint]

  • Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus non-structural protein (nsp) 11 was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV) representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration-dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the USA and almost 1.5b in Europe every year. To find a way to combat these viruses, we focused on the essential viral non-structural protein (nsp) 11. Nsp11 is associated with multiple functions such as RNA-processing and suppressing the infected host innate immunity system. The three solved structures in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

  • The nonstructural protein NSs of Schmallenberg virus is targeted to the nucleolus and induces nucleolar disorganization. [PublishAheadOfPrint]

  • Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded non-structural protein, termed NSs, is a major virulence factor of SBV and it is known to promote the degradation of Rpb1, a subunit of the RNA Pol II complex, and therefore hampers global cellular transcription. In this study we found that NSs is mainly localized in the nucleus of infected cells, and, specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs co-localizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolar to nucleoplasm redistribution, evocative of viral-induced nucleolar disruption. In contrast, nucleolar pattern of B23 was unchanged upon infection with a SBV recombinant mutant with NSs lacking the NoLS motif (SBVNoLS). Interestingly, unlike wild type SBV, the inhibitory activity of SBVNoLS towards RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes cellular antiviral response and/or induces cell death.

    IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that has spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation with the central nervous system being one of the most affected organs. The viral-encoded NSs protein acts as a virulent factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for viral induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

  • Intrinsic and innate defenses of neurons: Detente with the herpesviruses. [PublishAheadOfPrint]

  • Neuroinvasive herpesviruses have evolved to efficiently infect and establish latency in neurons. The nervous system has limited capability to regenerate, so immune responses therein are carefully regulated to be non-destructive, with dependence on atypical intrinsic and innate defenses. In this article we review studies of some of these non-canonical defense pathways and how herpesvirus gene products counter them, highlighting the contributions that primary neuronal in vitro models have made to our understanding of this field.

  • The DNA damage signaling is required for replication of the HBoV1 DNA in dividing HEK293 cells [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1), an emerging human pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelia air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (PLoS Pathog., 2016; 12:e1005399), and HBoV1 replicates only in terminally differentiated, non-dividing cells. This report builds on the previous discovery that replication of the HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral non-structural protein NS1 is sufficient to induce the DDR and the activation of three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both replication of the HBoV1 DNA and downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA-replication factors and major DNA-repair DNA polymerases (Pol and Pol ) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of a DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest.

    IMPORTANCE The human parvovirus bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory-tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support replication of the duplex genome of this virus and produce progeny virions. In this study, we demonstrate that the HBoV1 induces a DDR that plays significant roles in replication of the viral DNA and production of progeny virions in HEK293 cells. We also show that both cellular DNA-replication factors and DNA-repair DNA polymerases colocalize within centers of viral DNA replication, and that Pol and Pol play an important role in HBoV1 DNA replication. Whereas the DDR that leads to replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 can activate all three PI3KKs (ATM, ATR, and DNA-PKcs).

  • High EBV load and genomic diversity are associated with generation of gp350 specific neutralizing antibodies following Acute Infectious Mononucleosis [PublishAheadOfPrint]

  • The Epstein Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis; AIM) and again 6 months later (convalescence, CONV). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at AIM and all 24 (100%) individuals at CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically-infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected in AIM (4 of 24 individuals; 17%) but was commonly detected in CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained 6 months post-diagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed inter-patient gp350 sequence variation, but conservation of the CR2 binding site. Levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels in AIM and greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM.

    IMPORTANCE Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles mediating uptake by immune cells or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro. The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains which may poorly reflect primary viral envelop diversity. We report some of the first primary gp350 sequences noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

  • In vivo efficacy of measles virus fusion protein-derived peptides is modulated by properties of self-assembly and membrane residence [PublishAheadOfPrint]

  • Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting into the target cell membrane and then re-folding into a post-fusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances bio-distribution and half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides.

    Importance Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F derived peptides.

  • Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry [PublishAheadOfPrint]

  • Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence/absence of protease inhibitors. We found that, when compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased such that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin.

    IMPORTANCE Many envelope viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease, TMPRSS2, rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases such as SARS and MERS should target cell surface TMPRSS2 rather than endosomal cathepsin.

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