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

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  • Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII [Cellular Response to Infection]

  • Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections.

    IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus- and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1.

  • Single-Dose Trivalent VesiculoVax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge [Vaccines and Antiviral Agents]

  • Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.

    IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.

  • Emergence of Double- and Triple-Gene Reassortant G1P[8] Rotaviruses Possessing a DS-1-Like Backbone after Rotavirus Vaccine Introduction in Malawi [Genetic Diversity and Evolution]

  • To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains (n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 x 10nndash;4 to 4.1 x 10nndash;3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.

    IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine escape mutants. While multiple African countries have introduced a rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the postvaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998 to 2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting a limited effect on the recognition of G1-specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation.

  • Respiratory Syncytial Virus: Targeting the G Protein Provides a New Approach for an Old Problem [Minireviews]

  • Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) annually affecting ggt;2 million children in the United States llt;5 years old. In the elderly (ggt;65 years old), RSV results in ~175,000 hospitalizations annually in the United States with a worldwide incidence of ~34 million. There is no approved RSV vaccine, and treatments are limited. Recently, a phase 3 trial in the elderly using a recombinant RSV F protein vaccine failed to meet its efficacy objectives, namely, prevention of moderate-to-severe RSV-associated LRTI and reduced incidence of acute respiratory disease. Moreover, a recent phase 3 trial evaluating suptavumab (REGN2222), an antibody to RSV F protein, did not meet its primary endpoint of preventing medically attended RSV infections in preterm infants. Despite these setbacks, numerous efforts targeting the RSV F protein with vaccines, antibodies, and small molecules continue based on the commercial success of a monoclonal antibody (MAb) against the RSV F protein (palivizumab). As the understanding of RSV biology has improved, the other major coat protein, the RSV G protein, has reemerged as an alternative target reflecting progress in understanding its roles in infecting bronchial epithelial cells and in altering the host immune response. In mouse models, a high-affinity, strain-independent human MAb to the RSV G protein has shown potent direct antiviral activity combined with the alleviation of virus-induced immune system effects that contribute to disease pathology. This MAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab.

  • The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation [Genome Replication and Regulation of Viral Gene Expression]

  • The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.

    IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

  • Effective Priming of Herpes Simplex Virus-Specific CD8+ T Cells In Vivo Does Not Require Infected Dendritic Cells [Virus-Cell Interactions]

  • Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection.

    IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo. Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.

  • Size, Composition, and Evolution of HIV DNA Populations during Early Antiretroviral Therapy and Intensification with Maraviroc [Genetic Diversity and Evolution]

  • Residual viremia is common during antiretroviral therapy (ART) and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial and monitored for a median of 107 weeks. Participants started ART with (n = 9) or without (n = 9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 env, gag, and pol (454 Roche) was performed on longitudinally collected plasma and peripheral blood mononuclear cell (PBMC) samples while on ART. Sequence data were analyzed for evidence of evolution by (i) molecular diversity analysis, (ii) nonparametric test for panmixia, and (iii) tip date randomization within a Bayesian framework. There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (nndash;0.08 pplusmn; 0.01 versus nndash;0.09 pplusmn; 0.01 log10 copies/week in MVC+ versus MVCnndash; groups; P = 0.62). All participants had low-level residual viremia (median, 2.8 RNA copies/ml). Across participants, medians of 56 (interquartile range [IQR], 36 to 74), 29 (IQR, 25 to 35), and 40 (IQR, 31 to 54) haplotypes were generated for env, gag, and pol regions, respectively. There was no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed, and potential low-level viral replication should always be considered in cure strategies.

    IMPORTANCE Residual viremia is common among HIV-infected people on ART. It remains controversial if this viremia is a consequence of propagating infection. We hypothesized that molecular evolution would be detectable during viral propagation and that therapy intensified with the entry inhibitor maraviroc would demonstrate less evolution. We performed a randomized double-blinded treatment trial with 18 acutely infected men (standard ART versus standard ART plus maraviroc). From longitudinally collected blood plasma and cells, levels of HIV DNA and cell-free HIV RNA were quantified by droplet digital PCR, and HIV DNA (env, gag, and pol coding regions) was deep sequenced (454 Roche). Investigating people who started ART during the earliest stages of their HIV infection, when viral diversity is low, provides an opportunity to detect evidence of viral evolution. Despite using a battery of analytical techniques, no clear and consistent evidence of viral propagation for over 90 weeks of observation could be discerned.

  • Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection [Cellular Response to Infection]

  • Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.

    IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

  • Peripheral Blood Biomarkers of Disease Outcome in a Monkey Model of Rift Valley Fever Encephalitis [Pathogenesis and Immunity]

  • Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8+ T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4+ and CD8+ T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-aalpha;) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model.

    IMPORTANCE Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.

  • Truncating the gp41 Cytoplasmic Tail of Simian Immunodeficiency Virus Decreases Sensitivity to Neutralizing Antibodies without Increasing the Envelope Content of Virions [Pathogenesis and Immunity]

  • An incomplete understanding of native human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins (Envs) impedes the development of structural models of Env and vaccine design. This shortcoming is due in part to the low number of Env trimers on virus particles. For SIV, this low expression level can be counteracted by truncating the cytoplasmic tail (CT) of Env. CT truncation has been shown to increase Env incorporation into the virion and is commonly used in vaccine and imaging studies, but its effects on viral antigenicity have not been fully elucidated. To study the effects of a CT truncation of Env in viruses in similar genetic contexts, we introduced stop codons into the CT of a SIVsmE660 molecular clone and two neutralizing antibody (NAb) escape variants. These viruses shared 98% sequence identity in Env but were characterized as either tier 1 (sensitive to neutralization), tier 2 (moderately resistant to neutralization), or tier 3 (resistant to neutralization). However, the introduction of premature stop codons in Env at position Q741/Q742 converted all three transfection-derived viruses to a tier 3-like phenotype, and these viruses were uniformly resistant to neutralization by sera from infected macaques and monoclonal antibodies (MAbs). These changes in neutralization sensitivity were not accompanied by an increase in either the virion Env content of infection-derived viruses or the infectivity of transfection-derived viruses in human cells, suggesting that CT mutations may result in global changes to the Env conformation. Our results demonstrate that some CT truncations can affect viral antigenicity and, as such, may not be suitable surrogate models of native HIV/SIV Env.

    IMPORTANCE Modifications to the SIV envelope protein (Env) are commonly used in structural and vaccine studies to stabilize and increase the expression of Env, often without consideration of effects on antigenicity. One such widespread modification is the truncation of the Env C-terminal tail. Here, we studied the effects of a particular cytoplasmic tail truncation in three SIVsm strains that have highly similar Env sequences but exhibit different sensitivities to neutralizing antibodies. After truncation of the Env CT, these viruses were all very resistant to neutralization by sera from infected macaques and monoclonal antibodies. The viruses with a truncated Env CT also did not exhibit the desired and typical increase in Env expression. These results underscore the importance of carefully evaluating the use of truncated Env as a model in HIV/SIV vaccine and imaging studies and of the continued need to find better models of native Env that contain fewer modifications.

  • Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress [Structure and Assembly]

  • During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV.

    IMPORTANCE Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus, where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV, but not those found in ODV, using a virus-encoded ubiquitin (vUbi). Therefore, ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV, thus ensuring lethal infection of the host.

  • Adaptive Immune Responses following Senecavirus A Infection in Pigs [Pathogenesis and Immunity]

  • Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of aalpha;bbeta; T cells, especially CD4+ T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8+ and double-positive CD4+ CD8+ T cells (effector/memory T cells) expressing interferon gamma (IFN-) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host.

    IMPORTANCE Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4+ T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.

  • ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment [Vaccines and Antiviral Agents]

  • Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8+ T and NK cell populations in vitro. Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8+ T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8+ effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ~2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common C and bbeta; chain receptors on both CD8+ T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8+ T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs.

    IMPORTANCE Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen.

  • Flow Virometry: a Powerful Tool To Functionally Characterize Viruses [Minireviews]

  • For several decades, flow cytometry has been a common approach to analyze cells and sort them to near-purity. It enables one to probe inner cellular molecules, surface receptors, or infected cells. However, the analysis of smaller entities such as viruses and exocytic vesicles has been more difficult but is becoming mainstream. This has in part been due to the development of new instrumentation with resolutions below that of conventional cytometers. It is also attributed to the several means employed to fluorescently label viruses, hence enabling them to stand out from similarly sized particles representing background noise. Thus far, more than a dozen different viruses ranging in size from 40 nm to giant viruses have been probed by this approach, which was recently dubbed "flow virometry." These studies have collectively highlighted the breadth of the applications of this method, which, for example, has elucidated the maturation of dengue virus, served as quality control for vaccinia vaccines, and enabled the sorting of herpes simplex virus discrete viral particles. The present review focuses on the means employed to characterize and sort viruses by this powerful technology and on the emerging uses of flow virometry. It similarly addresses some of its current challenges and limitations.

  • A Mechanism for Priming and Realignment during Influenza A Virus Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3' terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.

    IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

  • Initiation, Elongation, and Realignment during Influenza Virus mRNA Synthesis [Genome Replication and Regulation of Viral Gene Expression]

  • The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.

    IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.

  • Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity [Structure and Assembly]

  • Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.

    IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

  • Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4+ and CD8+ T Lymphocytes In Vitro [Virus-Cell Interactions]

  • Accumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infect in vitro two closely related but functionally distinct immune cell types, CD4+ and CD8+ T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4+ or CD8+ T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4+ than in CD8+ T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4+ and CD8+ cells at estimated mean levels pplusmn; standard errors of the means of 6.7 x 102 pplusmn; 3.8 x 102 and 1.2 x 102 pplusmn; 0.8 x 102 copies/mmu;g RNA, respectively (P llt; 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4+ and in 1.2% of CD8+ T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4+ and CD8+ cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.

    IMPORTANCE Although the liver is the main site of HCV replication, infection of the immune system is an intrinsic characteristic of this virus independent of whether infection is symptomatic or clinically silent. Many fundamental aspects of HCV lymphotropism remain uncertain, including the degree to which different immune cells support infection and contribute to virus diversity. We show that authentic, patient-derived HCV productively replicates in vitro in two closely related but functionally distinct types of T lymphocytes, CD4+ and CD8+ cells. The display of viral proteins and unique variants, the production of virions with biophysical properties distinct from those in plasma serving as inocula, and inhibition of replication by an antiviral agent led us to ascertain that both T cell subtypes supported virus propagation. Infection of CD4+ and CD8+ T cells, which are central to adaptive antiviral immune responses, can directly affect HCV clearance, favor virus persistence, and decisively influence the development and progression of hepatitis C.

  • Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein [Virus-Cell Interactions]

  • The papillomavirus (PV) E2 protein is a sequence-specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Posttranslational modifications (PTMs) of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine 111 (K111) as a target of p300 acetylation in bovine PV (BPV). The di-lysines at 111 and 112 are conserved in almost all papillomaviruses. We pursued a mutational approach to query the functional significance of lysine in human PV (HPV) E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1-mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. While the replication-defective E2-K111R mutant recruited E1 to the viral replication origin, surprisingly, unwinding of the duplex DNA did not occur. In contrast, the K111 glutamine (K111Q) mutant increased origin melting and stimulated replication compared to wild-type E2. These experiments reveal a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.

    IMPORTANCE HPV is one of the most common sexually transmitted infections in the United States. Over 200 HPVs have been described, and they manifest in a variety of ways; they can be asymptomatic or can result in benign lesions (papillomas) or progress to malignancy. Although 90% of infections are asymptomatic and resolve easily, HPV16 and -18 alone are responsible for 70% of all cervical cancers, which are almost entirely caused by HPV infection. Interestingly, 60 to 90% of other cancers have been linked to HPV. The goal of this research is to further elucidate the mechanisms that regulate and mediate viral replication.

  • Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.

    IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.

  • Entry of Human Coronavirus NL63 into the Cell [Virus-Cell Interactions]

  • The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.

    IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

  • Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles [Vaccines and Antiviral Agents]

  • Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.

    IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

  • {beta}-D-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome [Vaccines and Antiviral Agents]

  • Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, bbeta;-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections.

    IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that bbeta;-d-N4-hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 mmu;M. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.

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

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    JVI Accepts: Articles Published Ahead of Print

  • Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques [PublishAheadOfPrint]

  • Simian varicella virus (SVV), the primate counterpart of varicella zoster virus, causes varicella (chickenpox), establishes latency in ganglia and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing the enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and infected monkeys. Herein, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo. One harbors eGFP fused to the N-terminus of open reading frame (ORF) 9 (rSVV.eGFP-2a-ORF9) and another in which eGFP was fused to the C-terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike the SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (IT) or IT plus intravenous (IV) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) at 10 days post-infection (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.

    IMPORTANCE SVV infection in non-human primates has served as an extremely useful animal model to study VZV pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

  • Novel Human Polyomavirus non-coding control regions differ in bi-directional gene expression according to host cell, large T-antigen expression, and clinically occurring rearrangements [PublishAheadOfPrint]

  • Human polyomavirus (HPyV) DNA genomes contain three regions denoted early viral gene region (EVGR) encoding the regulatory T-antigens and one microRNA, late viral gene region (LVGR) encoding the structural Vp capsid proteins, and non-coding control region (NCCR). The NCCR harbours the origin of viral genome replication and bi-directional promoter/enhancer functions governing EVGR- and LVGR-expression on opposite DNA strands. Despite principle similarities, HPyV-NCCRs differ in length, sequence, and architecture. To functionally compare HPyV-NCCRs, sequences from human isolates were inserted into a bi-directional reporter vector using dsRed2 for EVGR- and GFP for LVGR-expression, respectively. Transfecting HPyV-NCCR reporter vectors into human embryonic kidney (HEK)-293 cells and flow cytometry normalized to archetype BKPyV-NCCR revealed a hierarchy of EVGR-expression levels with MCPyV-, HPyV12, and STLPyV-NCCRs conferring stronger, and HPyV6-, HPyV9-, and HPyV10-NCCRs weaker levels, while LVGR-expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing SV40-large T-antigen (LTag) increased EVGR-expression for most HPyV-NCCRs, which correlated with the number of LTag-binding sites (Spearman r 0.625; pllt;0.05) and decreased following SV40-LTag siRNA knock-down. LTag-dependent activation was specifically confirmed for two different MCPyV-NCCRs in 293MCT cells expressing the cognate MCPyV-LTag. HPyV-NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV-NCCR activity, which partly synergized with SV40-LTag expression. Clinically occurring NCCR-sequence rearrangements of HPyV7-PITT-1 and -2, and HPyV9-UF1 were found to increase EVGR-expression compared to the respective HPyV archetype, but which was partly host cell type-specific.

    Author's Summary HPyV-NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV-NCCRs differ not only in sequence length, number, and position of LTag- and common transcription factor-binding sites, but also confer differences in bi-directional viral gene expression. Importantly, EVGR-reporter expression was significantly modulated by LTag-expression and by host cell properties. Clinical sequence variants of HPyV7- and HPyV9-NCCRs containing deletions and insertions were associated with increased EVGR-expression similar to BKPyV- and JCPyV-rearrangements emphasizing that HPyV-NCCR sequences are major determinants not only of host cell tropism, but also pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop pre-clinical models of HPyV persistence and replication, and suitable antiviral targets.

  • An alternate route for adeno-associated virus entry independent of AAVR [PublishAheadOfPrint]

  • Determinants and mechanisms of cell attachment and entry steer the Adeno-Associated Virus (AAV) in its utility as a gene therapy vector. Thus far a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of multiplicity of infection. Viral overlay assays and rescue experiments in non-permisive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knock out mice were equally permissive to transduction by AAVrh32.33 compared to wild type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multi-modal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved amongst all primate AAVs except for those in the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of entry of AAV, a vector system of broad utility in gene therapy.

    Importance: Adeno-Associated Virus (AAV) is a non-pathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies of this vector.

  • Gamma-herpesvirus colonization of the spleen requires lytic replication in B cells [PublishAheadOfPrint]

  • Gamma-herpesviruses infect lymphocytes and cause lymphocytic cancers. Murid Herpesvirus-4 (MuHV-4), Epstein-Barr virus and the Kaposi's Sarcoma-associated Herpesvirus all infect B cells. Latent infection can spread by B cell recirculation and proliferation, but whether this alone achieves systemic infection is unclear. To test the need of MuHV-4 for lytic infection in B cells we flanked its essential ORF50 lytic transactivator with loxP sites, then infected mice with B cell-specific cre expression (CD19-cre). The floxed virus replicated normally in cre- mice. In CD19-cre mice, nasal and lymph node infections were maintained but there was little splenomegaly and splenic virus loads remained low. Cre-mediated removal of other essential lytic genes gave a similar phenotype. CD19-cre spleen infection by intraperitoneal virus was also impaired. Therefore MuHV-4 had to emerge lytically from B cells to colonize the spleen. An important role for B cell lytic infection in host colonization is consistent with the large CD8+ T cell responses made to gamma-herpesvirus lytic antigens during infectious mononucleosis, and suggests that vaccine-induced immunity capable of suppressing B cell lytic infection might reduce long-term virus loads.

    IMPORTANCE Gamma-herpesviruses cause B cell cancers. Most models of host colonization derive from cell cultures with continuous, virus-driven B cell proliferation. However vaccines based on these models have worked poorly. To test whether proliferating B cells suffice for host colonization, we inactivated the capacity of MuHV-4, a gamma-herpesvirus of mice, to re-emerge from B cells. The modified virus was able to colonize a first wave of B cells in lymph nodes, but spread poorly to B cells in secondary sites such as the spleen. Consequently viral loads remained low. These results were consistent with virus-driven B cell proliferation exploiting normal host pathways, and so having to transfer lytically to new B cells for new proliferation. We conclude that viral lytic infection is a potential target to reduce B cell proliferation.

  • Myxoma Virus M083 is a virulence factor which mediates systemic dissemination [PublishAheadOfPrint]

  • Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro, their specific roles during the pathogenesis of poxviral disease remains largely unclear. Here we present data demonstrating that the putative chondroitin binding protein, M083, from the leporipoxvirus myxoma is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection mediated mortality. In vitro, removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate spread of myxoma onto rabbit lymphocytes allowing these cells to disseminate virus throughout infected rabbits.

    IMPORTANCE Poxviruses represent both a class of human pathogens as well as potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro, how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes.

  • Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles [PublishAheadOfPrint]

  • Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope allowing early gene expression, and breaching of the core wall allowing DNA release, replication and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68 kDa ankyrin-repeat/F box protein (68k-ank), associated with the cellular SCF ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the formation of DNA pre-replication sites and degradation of viral cores were severely diminished indicating that the 68k-ank deletion mutant had a defect in genome uncoating as well as an additional independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect suggesting the existence of compensating genes. By inserting VACV genes into a MVA 68k-ank deletion mutant, we discovered that M2, a member of the Poxvirus Immune Evasion (PIE) domain superfamily and a regulator of NF-B, and that C5, a member of the BTB/kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.

    IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that anyone of three seemingly unrelated and individually non-essential proteins are required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.

  • Porcine Mx1 protein inhibits classical swine fever virus replication by targeting nonstructural protein NS5B [PublishAheadOfPrint]

  • Mx proteins are interferon-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-CSFV activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro. siRNA experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon-aalpha; (IFNaalpha;) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase of CSFV. We used mutations in the poMX1 protein to elucidate the mechanism of their anti-CSFV activity and found that those that disrupted the association with NS5B, lost all anti-CSV activity. Moreover, RNA-dependent RNA polymerase (RdRp) activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Taken together, porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B.

    Importance: Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo, but the molecular mechanism of action remains largely unknown. In this study we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro. Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMX1 further elucidate the mechanism of their anti-CSFV activities.

  • The 5' Untranslated Region of the Major Immediate Early mRNA is Necessary for Efficient Human Cytomegalovirus Replication [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) IE1 and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the post-transcriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5' untranslated region (5' UTR) plays an important role in regulating mRNA translation. Therefore to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared major immediate early (MIE) transcripts IE1 and IE2 5' UTR (MIE 5' UTR) in regulating translation. In a cell-free system the MIE 5' UTR repressed translation, as predicted based on its length and sequence composition. However in transfected cells we found that the MIE 5' UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5rrsquo; UTR has a positive role in translation control. We also found that the MIE 5' UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5' UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA, and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild type MIE 5' UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5' UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.

    IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. Thus defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5' UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression, and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation, and thus inhibit lytic replication or prevent HCMV reactivation.

  • HIV replication and latency in a humanized NSG mouse model during suppressive oral combinational ART [PublishAheadOfPrint]

  • Although current combinatorial antiretroviral therapy (cART) is therapeutically effective in the majority of HIV patients, interruption of therapy can cause a rapid rebound in viremia, demonstrating the existence of a stable reservoir of latently infected cells. HIV latency is therefore considered a primary barrier to HIV eradication. Identifying, quantifying, and purging the HIV reservoir is crucial to effectively curing patients and relieving them from the lifelong requirement for therapy. Latently infected transformed cell models have been used to investigate HIV latency; however, they cannot accurately represent the quiescent cellular environment of primary latently infected cells in vivo. For this reason, in vivo humanized murine models have been developed for screening antiviral agents, identifying latently infected T-cells, and establishing treatment approaches for HIV research. Such models include humanized bone marrow/liver/thymus (BLT) mice and SCID-hu-thy/liv mice, which are repopulated with human immune cells and implanted human tissues through laborious surgical manipulation. However, no one has utilized the human hematopoietic stem cell (HSC)-engrafted NOD/SCID/IL2rnull (NSG) model (hu-NSG) for this purpose. Therefore, in the present study we used the HIV-infected hu-NSG mouse to recapitulate the key aspects of HIV infection and pathogenesis in vivo. Moreover, we evaluated the ability of HIV-infected human cells isolated from HIV-infected hu-NSG mice on suppressive cART to act as a latent HIV reservoir. Our results demonstrate that the hu-NSG model is an effective surgery-free in vivo system in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions.

    IMPORTANCE HIV can establish a stably integrated, non-productive state of infection at the level of individual cells, known as HIV latency, which is considered a primary barrier to curing HIV. A complete understanding of the establishment and role of HIV latency in vivo would greatly enhance attempts to develop novel HIV purging strategies. An ideal animal model for this purpose should be easy to work with, should have a shortened disease course so that efficacy testing can be completed in a reasonable time, and should have immune correlates that are easily translatable to humans. We therefore describe a novel application of the HSC-transplanted humanized NSG model for dynamically testing antiretroviral treatment, supporting HIV infection, establishing HIV latency in vivo. The hu-NSG model could be a facile alternative to humanized BLT or SCID-hu-thy/liv mice in which laborious surgical manipulation and time-consuming human cell reconstitution is required.

  • A new quinoline BRD4 inhibitor targets a distinct latent HIV-1 reservoir for re-activation from other 'shock drugs [PublishAheadOfPrint]

  • Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol) that is able to reactivate viral transcription in several models of HIV latency including J-Lat cells through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or llsquo;shockrrsquo; drugs such as PKC agonists or HDAC inhibitors. Here we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a distinct subset of latently integrated barcoded proviruses from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of the BET bromodomain inhibitors, which due to its minimalistic structure holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV.

    IMPORTANCE The tentative "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. Yet to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a "shock" regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo.

    Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators, HDAC and bromodomain inhibitors all target different subsets of proviral integrations. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory "shock" cocktail therapies for an improved reservoir reactivation.

  • Human Herpesvirus 8 Interferon Regulatory Factors 1 and 3 Mediate Replication and Latency Activities via Interactions with USP7 Deubiquitinase [PublishAheadOfPrint]

  • Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs 1-4) that likely function to suppress innate immune and cellular stress responses through inhibitory interactions with various cellular proteins involved in these activities. It is notable that vIRFs 1 and 4 have been reported to interact with the deubiquitinase USP7, substrates of which include p53 and p53-targeting and destabilizing ubiquitin E3 ligase MDM2. Structural studies of vIRF-1 and vIRF-4 USP7-binding sequences in association with USP7 have been reported; both involve interactions with N-terminal domain residues of USP7, via EGPS and ASTS motifs in vIRF-1 and vIRF-4, respectively, but vIRF-4 residues also contact the catalytic site. However, the biological activities of vIRF-1 and vIRF-4 via USP7 interactions are unknown. Here, we report that vIRF-3, which is latently as well as lytically expressed in HHV-8-infected primary effusion lymphoma (PEL) cells, also interacts with USP7, via duplicated EGPS motifs, and that this interaction is important for PEL cell growth and viability. The interaction also contributes to suppression of productive virus replication by vIRF-3, which we identify here. We further show that vIRF-1, which is expressed at low levels in PEL latency, promotes latent PEL cell viability, and that this activity and vIRF-1-promoted productive replication (reported previously) involve EGPS motif-mediated USP7 targeting by vIRF-1. This study is the first to identify latent and lytic functions of vIRF-1 and vIRF-3, respectively, and to address the biological activities of these vIRFs, in infected cells, through their interactions with USP7.

    IMPORTANCE HHV-8 is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease; both latent and lytic viral functions are believed to contribute. Viral interferon regulatory factors specified by HHV-8 are thought to be critically important for successful productive replication, through suppression of innate immune and stress responses triggered by the lytic cycle. Latently expressed vIRF-3 contributes significantly to PEL cell survival. Here, we identify ubiquitin-specific protease 7 (USP7) deubiquitinase targeting by vIRF-3 (in addition to previously reported USP7 binding by vIRF-1 and vIRF-4), the importance of vIRF-1 and vIRF-3 interactions with USP7 for latent PEL cell growth and viability, and the positive and negative contributions, respectively, of USP7 targeting by vIRF-1 and vIRF-3 to HHV-8 productive replication. This is the first report of the biological importance of vIRF-1 in PEL latency, the modulation of productive replication by vIRF-3, and the contributions of vIRF-USP7 interactions to HHV-8 biology.

  • Roles of the 5' untranslated region of non-primate hepacivirus in translation initiation and viral replication. [PublishAheadOfPrint]

  • The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III and IV) and a pseudoknot, is essential for translation and viral replication. Equine non-primate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5' -terminal domain (I), three additional domains (I', II and III), which are homologous to domains I, II and III, respectively, of HCV, and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domain I to I' of EHcV (5' proximal EHcV-specific region) improved RNA stability and provided HCV SGR with miR122-independent replication capability, while the EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domain I to I' improves RNA stability and viral replication regardless of miR-122 expression. The 5' proximal EHcV-specific region may exert an inherent mechanism to facilitate viral replication in non-hepatic tissues.

    IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and a liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs of horses, donkeys and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5' proximal EHcV-specific region enhances viral replication and RNA stability in an miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

  • Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA Damaging Agents: Implications for Oncolytic Virus Therapy [PublishAheadOfPrint]

  • We have previously shown that the Parainfluenza virus 5 (PIV5) mutant P/V-CPI- is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burden in mouse model systems. Here we show that P/V-CPI- infection of human laryngeal cancer HEp-2 cells results in the majority of the cells dying, but unexpectedly, over time there is an emergence of a population of cells which survive as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on activity of cellular inhibitors of apoptosis (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, the PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to inhibition of wild-type p53 inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. Similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI-, as well as acute infections with WT PIV5 and the related human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors which cannot establish PI, as well as the potential for combining chemotherapy with oncolytic RNA virus vectors.

    IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells which have gained resistance to chemotherapies. We have found that infection with the cytoplasmic-replicating Parainfluenza virus can result in increases in killing of cancer cells by agents which induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors which cannot establish a persistent infection, the repurposing of drugs that target cellular IAPs as antivirals and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors.

  • Increased epitope complexity correlated with antibody affinity maturation and a novel binding mode revealed by structures of rabbit antibodies against the third variable loop (V3) of HIV-1 gp120 [PublishAheadOfPrint]

  • The V3 loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (mAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these mAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (303TRKSIHIGPGRAF317), using the cradle binding mode similar to human V3 mAbs encoded by IGHV5-51 germline genes and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (308HIGPGRAFYTTGEI323). Unexpectedly, the 315RAFYTT320 portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity from human antibody germline genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.

    IMPORTANCE The apex crown of the third variable loop (V3) of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein and many mAbs targeting this region have been developed. Structural understanding of V3 crown mAbs not only can help understand how antibody responses targeting this unique region, but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 mAbs, 10A3 and 10A37, developed from rabbits immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.

  • Structural study of the C-terminal domain of non-structural protein 1 from Japanese encephalitis virus [PublishAheadOfPrint]

  • Japanese encephalitis virus (JEV) is a mosquito-transmitted Flavivirus that is closely related to other emerging viral pathogens including dengue, West Nile (WNV) and Zika viruses. JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific islands and the Far East, causing around 68,000 cases worldwide each year. In this study, we present a 2.1 AAring; resolution crystal structure of the C-terminal bbeta;-ladder domain of JEV non-structural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to WNV and ZIKV but differs form DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the monoclonal antibody, 22NS1, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes, and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This study highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships.

    IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Non-structural protein 1 (NS1) plays a role in viral replication and, because it is secreted, it can exhibit a wide range of interations with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the the C-terminal bbeta;-ladder domain of NS1 between flaviviruses including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics.

  • High-Resolution Sequencing of Viral Populations During Early SIV Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses [PublishAheadOfPrint]

  • Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (lle; 29 weeks post-infection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultra-deep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.

    IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep sequencing-based fitness assay we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

  • Cellular and Humoral Immunity Protect against Vaginal Zika Virus Infection in Mice [PublishAheadOfPrint]

  • Zika virus (ZIKV), which can cause devastating disease in fetuses of infected pregnant women, can be transmitted by mosquito inoculation and sexual routes. Little is known about immune protection against sexually transmitted ZIKV. In this study, we show that previous infection through intravaginal or subcutaneous routes with a contemporary Brazilian strain of ZIKV can protect against subsequent intravaginal challenge with a homologous strain. Both routes of inoculation induced high titers of ZIKV-specific and neutralizing antibody in serum and the vaginal lumen. Virus-specific T cells were recruited to and retained in the female reproductive tract after intravaginal and subcutaneous ZIKV infection. Studies in mice with genetic or acquired deficiencies in B and/or T cells demonstrated that both lymphocyte populations redundantly protect against intravaginal challenge in ZIKV-immune animals. Passive transfer of ZIKV immune IgG or T cells significantly limited intravaginal infection of naïve mice, although antibody more effectively prevented dissemination throughout the reproductive tract. Collectively, our experiments begin to establish the immune correlates of protection against intravaginal ZIKV infection, which should inform vaccination strategies in non-pregnant and pregnant women.

    IMPORTANCE The recent ZIKV epidemic resulted in devastating outcomes in fetuses and may affect reproductive health. Unlike other flaviviruses, ZIKV can be spread by sexual contact as well as a mosquito vector. While previous studies have identified correlates of protection for mosquito-mediated infection, few have focused on immunity against sexual transmission. As exposure to ZIKV via mosquito bite has likely occurred to many living in endemic areas, our study addresses whether this route of infection can protect against subsequent sexual exposure. We demonstrate that subcutaneous ZIKV infection can protect against subsequent vaginal infection by generating both local antiviral T cell and antibody responses. Our research begins to define the immune correlates of protection for ZIKV infection in the vagina and provides a foundation for testing ZIKV vaccines against sexual transmission.

  • Advancing our understanding of protective maternal immunity as a guide for development of vaccines to reduce congenital cytomegalovirus infections [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is the most common congenitally transmitted pathogen worldwide, impacting an estimated 1 million newborns annually. Congenital HCMV (cCMV) infection is a major global contributor to long-term neurologic deficits, including deafness, microcephaly, neurodevelopmental delay, as well as fetal loss and occasional infant mortality. Accordingly, a maternal vaccine to prevent cCMV continues to be a top public health priority. Nevertheless, we remain without a licensed vaccine. Maternal immunity provides partial protection, as the risk of vertical HCMV transmission from chronically infected mothers is reduced compared to settings in which the mother is newly infected during pregnancy. Therefore, an understanding of the maternal immune correlates of protection against cCMV are critical to informing design of an efficacious maternal vaccine. Although vaccine development is being assiduously pursued by a large number of pharmaceutical manufacturers, biotechnology organizations, and academic researchers, some pessimism has been expressed regarding the question of whether a vaccine to protect against cCMV is possible. This pessimism is based on observations that natural immunity is not completely protective against maternal reinfection and congenital transmission. However, we assert that optimism regarding vaccine development is indeed justified, based on accruing evidence of immune correlates of protection mmdash; readily achievable by vaccination mmdash; that are associated with reduced transmission of HCMV to the fetus in seronegative women. In light of the substantial burden on society conferred by cCMV infection, even a modest reduction of this fetal disease is an important public health goal, and justifies aggressive clinical evaluation of vaccines currently in the pipeline.

  • Casein kinase 1{alpha} mediates degradation of receptors for type I and type II interferons caused by hemagglutinin of influenza A virus [PublishAheadOfPrint]

  • Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-) receptor 1 (IFNGR1) as well as IFNAR1 via casein kinase 1aalpha; (CK1aalpha;), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN--stimulated genes in response to exogenously supplied recombinant IFN-. Importantly, CK1aalpha;, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1aalpha; or siRNA-based knockdown of CK1aalpha; repressed the degradation process of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1aalpha; was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1aalpha;, creating a condition favorable for viral propagation. Therefore, the study uncovers a new immune evasive pathway of influenza virus.

    IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans by causing seasonal and pandemic influenza. Upon infection, the innate and adaptive immunity such as the interferon (IFN) response is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses. Yet, the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of type II IFN receptor, IFNGR1 and thereby substantially attenuates cellular responses to IFN-. Of note, a cellular kinase, casein kinase 1aalpha; (CK1aalpha;) is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1aalpha; is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may cast new insights into the interplay between IAV and host immunity to impact influenza pathogenicity.

  • Non-structural protein {sigma}1s is required for optimal reovirus protein expression [PublishAheadOfPrint]

  • Reovirus non-structural protein 1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of the 1s protein during the reovirus replication cycle is not known. In this study, we found that 1s was required for efficient reovirus replication in SV40-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild type reovirus produced substantially higher viral titers than a 1s-deficient mutant. The 1s protein was not required for early events in the reovirus infection, as no difference in infectivity between the wild type and 1s-null viruses was observed. However, wild type virus produced markedly higher viral protein levels than the 1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the 1s protein was dispensable for cell killing and induction of type-1 interferon responses. In the absence of 1s, viral factory (VF) maturation was impaired, but sufficient to support low levels of reovirus replication. Together, our results indicate that 1s is not absolutely essential for viral protein production, but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that 1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.


    Hematogenous dissemination is critical a step in the pathogenesis of many viruses. For reovirus, nonstructural protein 1s is required for viral spread via the blood. However, the mechanism by 1s promotes reovirus dissemination is unknown. Here, we identified 1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which 1s is required for efficient reovirus replication. In these cells, wild type virus produced substantially higher levels of viral protein than a 1s-deficient mutant. The 1s protein was not required for viral mRNA transcription or viral protein stability. Owing to reduced levels of viral protein synthesized in the absence of 1s, maturation of viral factories was impaired and significantly fewer viral progeny were produced. Taken together, our findings indicate that 1s is required for optimal reovirus protein production, and thereby viral replication, in cells required hematogenous reovirus dissemination.

  • Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus [PublishAheadOfPrint]

  • Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North-American type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in non-structural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss in virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 were involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus.


    Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in a HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity, and also provide new avenues of research for the study of other viruses.

  • Infection and replication of influenza virus at the ocular surface [PublishAheadOfPrint]

  • Though influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subject to aerosol inoculation. The temperature at which cells were cultured post-inoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33ddeg;C relative to 37ddeg;C and this effect was slightly greater for the conjunctivitis isolates than the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multi-layer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited hemagglutination of both ocular and non-ocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of non-ocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells, but that this is more likely for ocular tropic viruses as they are better able to maintain their infectivity during passage through the tear film.

    IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza viral tropism have yet to be fully elucidated. Here, we sought to identify factors which limit the ability of most influenza viruses to cause ocular infection. Though ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors which influence infection and replication in this non-canonical site may point toward novel determinants of tropism in the respiratory tract.

  • Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA [PublishAheadOfPrint]

  • KSHV lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. Virus-encoded b-Zip family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here we report that K8 is a RNA binding protein, which also associates with many proteins including other RNA binding proteins. Many K8-involved protein-protein interactions are mediated by RNA. Using a crosslinking and immunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, that include both viral (PAN, T1.4, T0.7 and etc.) and cellular (MALAT-1, MRP, 7SK and etc.) RNAs. An RNA-binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 binding to many noncoding RNAs as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The function of K8 and associated T1.4 RNA was investigated in details and results showed that T1.4 mediates the binding of K8 with ori-Lyt DNA. T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble replication complex. Depletion of T1.4 abolished the DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection.


    Genome wide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding region are actually transcribed and give rise to stable RNAs. Emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes exampled by ribosome or spliceosome are not ancient relics of the last riboorganism but would be well adapted for regulatory role in biology. K8 has been puzzled by its unique characteristic such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study revealed the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiate DNA replication in coordination with a noncoding RNA. It is suggested that many of K8 functions, if not all, are carried out through its associated RNAs.

  • The Identification and Characterization of Sindbis Virus RNA:Host Protein Interactions [PublishAheadOfPrint]

  • Arthropod-borne viruses, such as the members of genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduces a robust viral RNA:protein discovery method to elucidate the Sindbis virus (SINV) RNA:Protein host interface. Cross-Link Assisted mRNP Purification (CLAMP) assessment reveals an extensive array of host/pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of Protein:vRNA interaction via a CLIP-seq approach and assessed the consequences of the RNA:protein binding event of hnRNP K, hnRNP I, and hnRNP M in regards to viral infection. Herein we demonstrate that mutation of the prioritized hnRNP:vRNA interaction sites effectively disrupted the hnRNP:vRNA interaction. Correlating with disrupted hnRNP:vRNA binding, SINV growth kinetics were reduced relative to wild type parental viral infections in a vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics were found to be dysregulated structural gene expression. Collectively, this study further defines the scope and importance of the alphavirus host/pathogen vRNA:protein interactions.

    IMPORTANCE Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their viral lifecycle. However, the extent, and importance, of host/pathogen RNA:protein interactions is woefully under characterized. The efforts detailed in this study fulfill this critical gap; and the significance of this research is three-fold. First, the data presented here fundamentally expands the scope and understanding of alphavirus host/pathogen interactions. Secondly, this study identifies the site of interactions for several prioritized interactions and defines the contribution of the RNA:protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of given host/pathogen interactions may be assessed, in the future, using a mouse model of infection.

  • HIV-1 Vif's capacity to manipulate the cell cycle is species-specific [PublishAheadOfPrint]

  • Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors Cyclin T1 (CCNT1) and Exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's post-integration stages, we studied HIV-1NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1 compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that the CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines that we mapped to the viral protein Vif and its apparent species-specific capacity to induce G2/M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that VifNL43's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G2/M cell cycle arrest in other non-human cell types, including cells derived from non-human primates, leading us to propose that one or more human-specific co-factors underpins Vif's ability to modulate the cell cycle.


    Cells derived from mice and other rodents exhibit profound blocks to human immunodeficiency virus type 1 (HIV-1) replication, thus hindering the development of alow cost small animal model for studying HIV/AIDS. Herein, we engineered otherwise non-permissive mouse cells to express HIV-1 compatible versions of two species-specific host dependency factors, Cyclin T1 (CCNT1) and Exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1 particle production but, unexpectedly, are completely resistant to virus-induced cytopathic effects. We mapped these effects to the viral accessory protein Vif that induces a prolonged G2/M cell cycle arrest followed by apoptosis in human cells. Combined, our results indicate that one or more additional human-specific co-factors govern HIV-1's capacity to modulate the cell cycle, with potential relevance to viral pathogenesis in people and existing animal models.

  • The E3 ubiquitin ligase Siah-1 suppresses avian reovirus infection by targeting p10 for degradation [PublishAheadOfPrint]

  • Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. Our previous report demonstrated that the cellular Lysosome Associated Membrane Protein (LAMP)-1 interacted with p10 and was involved in its degradation. However, the molecular mechanism underlying LAMP-1 mediated p10 degradation remains elusive. We report here that E3 ubiquitin ligase Seven in Absentia Homolog (Siah)-1 is critical for p10 ubiquitylation. Our data show that Siah-1 ubiquitylated p10 and targeted it for proteasome degradation. Furthermore, ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming multi-component complex. Thus, LAMP-1 promotes proteasomal degradation of p10 via interacting with both p10 and E3 ligase Siah-1. These data establish a novel host defense mechanism that LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection.

    IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. The ARV p10 protein is a virulence factor responsible for the induction of cell syncytium formation and apoptosis, and is rapidly degraded in host cells. We previously found that the cellular Lysosome Associated Membrane Protein (LAMP)-1 interacts with p10 and is involved in its degradation. Here we report that the E3 ubiquitin ligase Seven in Absentia Homolog (Siah)-1 ubiquitylated p10 and targeted it for proteasomal degradation. Furthermore, ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming multi-component complex. Thus, LAMP-1 serves as an adaptor to allow Siah-1 targeting p10 for degradation, thereby suppressing ARV growth in host cells.

  • Functional Analysis of the Dengue Virus Genome Using An Insertional Mutagenesis Screen [PublishAheadOfPrint]

  • In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness, and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genome wide transposon mutagenesis screen with Next Generation Sequencing to determine sites in the viral genome that tolerate 15 nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection, and found six of them capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.


    Dengue virus is a mosquito borne virus distributed in tropical and subtropical regions globally that can result in hospitalization and even death in some cases. Although a vaccine exists, its limitations and a lack of approved antiviral treatments highlight our limited understanding of dengue pathogenesis and host immunity. The functions of many viral proteins are poorly understood. We used a previously published approach using transposon mutagenesis to develop tools to study these proteins' functions by adding insertions randomly throughout the viral genomes. These genomes were transferred into cells and infectious progeny recovered to determine sites that tolerated insertions as only those genomes that tolerated insertions would be able to propagate. Using these results, we created viruses with epitope tags, one in the viral structural protein Capsid and one in the viral nonstructural protein NS4B. Further investigation of these mutants may elucidate the role of Capsid and NS4B during dengue infections.

  • Influenza C and D viruses package eight organized ribonucleoprotein complexes [PublishAheadOfPrint]

  • Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.

    IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

  • Germline IgM is sufficient, but not required, for antibody-mediated alphavirus clearance from the central nervous system [PublishAheadOfPrint]

  • Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase (AID)-/-, secretory IgM (sIgM)-/- and AID-/- sIgM-/- double knock out (DKO) mice with wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID-/- mice produce only germline IgM while sIgM-/- mice secrete IgG, but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM-/- mice, but disease severity, weight loss and survival were similar between groups. AID-/- mice produced high levels of SINV-specific IgM while sIgM-/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and amount of cell death in brain were comparable. We conclude that antibody is required and that either germline IgM or IgG is sufficient for clearance of virus from the CNS.

    IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain and either early germline IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral IgM is a determinant of outcome.

  • Glycosyl-phosphatidylinositol-Anchored Anti-HIV Env Single Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity [PublishAheadOfPrint]

  • In previous studies, we demonstrated that single-chain variable fragments (scFv) from anti-HIV Env monoclonal antibodies act as entry inhibitors when tethered to the surface of target cells by a glycosyl-phosphitidylinositol (GPI) anchor. Interestingly, even if a virus escapes inhibition at entry, its replication is ultimately controlled. We hypothesized that in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env in an infected cell, thereby interfering with infectivity of newly produced virions. Here, we show that expression of the anti-HIV Env GPI-scFvs in virus producing cells reduced the release of HIV from cells 5-22-fold, and infectivity of the virions that were released was inhibited by 74% to 99%. Additionally, anti-HIV Env GPI-scFv X5 inhibited virion production and infectivity after latency reactivation, and blocked transmitter/founder virus production and infectivity in primary CD4+ T cells. By contrast, SIV production and infectivity were not affected by the anti-HIV Env GPI-scFvs. Loss of infectivity of HIV was associated with a reduction in the amount of virion-associated Env gp120. Interestingly, an analysis of Env expression in cell lysates demonstrated that the anti-Env GPI-scFvs interfered with processing of Env gp160 precursors in cells. These data indicate that GPI-scFvs can inhibit Env processing and function, thereby restricting production and infectivity of newly synthesized HIV. Anti-Env GPI-scFvs therefore appear to be unique anti-HIV molecules, as they derive their potent inhibitory activity by interfering with both early (receptor binding/entry) and late (Env processing and incorporation into virions) stages of the HIV life cycle.

    Importance The restoration of immune function and persistence of CD4+ T cells in HIV-1 infected individuals without antiretroviral therapy requires a way to increase resistance of CD4+ T cells to infection by both R5- and X4-tropic HIV-1. Previously, we reported that anchoring anti-HIV-1 single chain variable fragments (scFvs) via glycosylphosphatidylinositol (GPI) to the surface of permissive cells conferred a high level of resistance to HIV-1 variants at the level of entry. Here, we report that anti-HIV GPI-scFvs also derive their potent antiviral activity in part by blocking HIV production and Env processing, which consequently, inhibits viral infectivity even in primary infection models. Thus, we conclude that GPI-anchored anti-HIV scFvs derive their potent blocking activity of HIV replication by interfering with successive stages of the viral life cycle. They may be effectively used in genetic intervention of HIV-1 infection.

  • Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation [PublishAheadOfPrint]

  • Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection, and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum-associated, IFN-inducible), which shows antiviral activity against a broad spectrum of viruses including several flaviviruses. Here we describe a novel antiviral mechanism exerted by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and co-localize with the structural proteins pre-membrane (prM) and envelope (E) of TBEV, as well as the non-structural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possibility of new drug targets that can be used for therapeutic intervention.


    Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism exerted by viperin to inhibit the replication of two flaviviruses; tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV non-structural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's anti-flaviviral activity. Taken together, our work shows important similarities and differences among the members of the genus Flavivirus, and could lead to the possibility of therapeutic intervention.

  • Mechanism of HIV-1 Resistance to an Electronically Constrained {alpha}-Helical Peptide Membrane Fusion Inhibitor [PublishAheadOfPrint]

  • SC29EK is an electronically constrained aalpha;-helical peptide HIV-1 fusion inhibitor highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (E43K/E49A) or three (Q39R/N43K/N126K, N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20, C34) and newly-designed (sifuvirtide, MT-SC29EK, 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair the ability of viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and inter-helical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights for the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.

    IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barrier for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained aalpha;-helical peptide, SC29EK is highly potent on both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first- and new-generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

  • Specific mutations in the PB2 protein of influenza A virus compensate for the lack of efficient IFN antagonism of the NS1 protein of bat influenza A-like viruses. [PublishAheadOfPrint]

  • Recently, two new influenza A-like viruses have been discovered in bats, HL17NL10 and HL18NL11. The hemagglutinin-like (HL) and neuraminidase like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A virus. We now have investigated whether the NS1 proteins of HL17NL10 and HL18NL11 viruses can functionally substitute the NS1 protein of a conventional influenza A virus. For this purpose we generated recombinant influenza A/PR8/34 (PR8) H1N1 viruses containing the NS1 protein of PR8 WT, HL17NL10 and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, r/NS1HL18) were tested for replication in bat and non-bat mammalian cells and in mice. Our results demonstrate that r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo. However, bat NS1 recombinant viruses showed similar phenotypes as r/NS1PR8 virus in STAT1-/- human A549 cells and mice, unable to respond to IFN. Interestingly, multiple mouse passages of r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, the selected PB2 mutants restored virulence and IFN antagonism. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than its conventional influenza A virus NS1 counterpart in antagonizing the IFN response, and that this deficiency can be overcome by the influenza virus PB2 protein.


    Significant gaps are still uncovered in our understanding of the basic features of the recently discovered bat influenza A-like viruses, HL17NL10 and HL18NL11. These unique viruses display both similarities and differences in basic biology compared to conventional influenza A viruses. In here, we show that recombinant influenza A viruses containing the NS1 protein from HL17NL10 and HL18NL11 are attenuated. This attenuation was mediated by their inability to antagonize the type I IFN response. However, this deficiency could be compensated by single amino acid replacements in the PB2 gene. Our results unravel a functional divergence between the NS1 proteins of bat influenza A-like and conventional influenza A viruses, and demonstrate an interplay between the viral PB2 and the NS1 protein to antagonize IFN.

  • MicroRNA-130a regulates both HCV and HBV replication through a central metabolic pathway [PublishAheadOfPrint]

  • BACKGROUND: HCV infection has been shown to regulate miR-130a in patient biopsies and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and HBV replication.

    METHODS: We used bioinformatics software including miRanda, TargetScan, PITA and RNAhybrid to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed, or knocked down by siRNA or by CRISPR/Cas9 gRNA, respectively. Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, JFH1 HCV-infected Huh7.5.1 cells and JFH1 HCV-infected primary human hepatocytes (PHHs), and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells and HBV-infected PHHs were measured by qRT-PCR and Western blot, respectively.

    RESULTS: We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (mimic) knocked down PKLR mRNA and protein levels. miR-130a inhibitor and gRNA increased PKLR expression, HCV replication and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication, and rescued the inhibition of HCV and HBV replication by miR-130a mimic and PKLR knockdown.

    CONCLUSION: miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provides novel insights into understanding key metabolic enzymatic pathway steps regulated by miR-130a, including PKLR and pyruvate, which are subverted by HCV and HBV replication.


    We identified that miR-130a regulates its target gene PKLR and its subsequent pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized miRNA regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral lifecycle and prevent establishment of persistent infection for HCV, HBV and, potentially, other viral infections.

  • Sustained specific and cross-reactive T cell responses to Zika and Dengue viruses NS3 in West Africa [PublishAheadOfPrint]

  • Recent studies on the role of T cells in Zika virus (ZIKV) infection have shown that T cell responses to Asian ZIKV infection are important for protection, and that previous Dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. Human T cell responses to African ZIKV infection, however, remain unexplored. Here, we utilized the modified anthrax toxin delivery system to develop a flavivirus ELISPOT. Using human ZIKV and DENV samples from Senegal, West Africa, our results demonstrate specific and cross-reactive T cell responses to nonstructural protein 3 (NS3). Specifically, we found that T cell responses to NS3 protease are ZIKV and DENV specific, but responses to NS3 helicase are cross-reactive. Sequential sample analyses revealed immune responses sustained many years after infection. These results have important implications for African ZIKV/DENV vaccine development, as well for potential flavivirus diagnostics based on T cell responses.


    The recent Zika virus (ZIKV) epidemic in Latin America and the associated congenital microcephaly and Guillain-Barreeacute; syndrome has raised questions as to why we have not recognized these distinct clinical diseases in Africa. The human immunologic response to ZIKV and related flaviviruses in Africa represents a research gap that may potentially shed light on the mechanisms contributing to protection. The goal of our study was to develop an inexpensive assay to detect and characterize the T cell response to African ZIKV and DENV. Our data show long-term specific and cross-reactive human immune responses against African ZIKV and DENV, suggesting the potential usefulness of a diagnostic based on the T cell response. Additionally, we show that prior flavivirus exposure influences the magnitude of the T cell response. The identification of immune responses to African ZIKV and DENV is of relevance to vaccine development.

  • Paramyxovirus V proteins interact with the RIG-I/TRIM25 regulatory complex and inhibit RIG-I signaling. [PublishAheadOfPrint]

  • Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both the RIG-I regulatory protein, TRIM25, and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem CARDs of RIG-I (the region of TRIM25-ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to MAVS. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism.

    IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with, and inhibit the activation of, RIG-I, thereby interrupting the antiviral signaling pathway, and facilitating virus replication.

  • The Wnt signaling pathway is differentially expressed during the bovine herpesvirus 1 latency-reactivation cycle: evidence that two protein kinases associated with neuronal survival (Akt3 and bone morphogenetic protein receptor 2) are expressed at higher levels during latency. [PublishAheadOfPrint]

  • Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with bbeta;-catenin and a bbeta;-catenin coactivator, high mobility group AT-hook 1 protein (HMGA1), which correlates with increased bbeta;-catenin dependent transcription and cell survival. bbeta;-catenin and HMGA1 are readily detected in a subset of latently infected TG neurons, but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/bbeta;-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/bbeta;-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to HSV-1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2 promotes latency.


    Life-long bovine herpesvirus 1 (BoHV-1) latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/bbeta;-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and bone morphogenetic protein receptor 2 (BMPR2), were expressed at higher levels during latency but repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and bbeta;-catenin dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival: processes crucial for life-long viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.

  • Therapeutic efficacy of vectored PGT121 gene delivery in HIV-1-infected humanized mice [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1 specific bNAb PGT121 in wildtype and immunocompromised C57Bl/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo. Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in greater reduction of viral loads as compared with AAV1.PGT121. Ad5.PGT121 also led to more sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of anti-antibody responses may also be required.


    Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1 specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice.

  • Digitoxin suppresses human cytomegalovirus replication via Na+, K+/ATPase {alpha}1 subunit-dependent AMPK and autophagy activation [PublishAheadOfPrint]

  • Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report on a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMPK activity and autophagy flux through the Na+, K+/ATPase aalpha;1 subunit. Our data illustrate an intricate balance between autophagy regulators AMPK, mTOR and ULK1 during infection and treatment with the cardiac glycoside, digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies of digitoxin and AICAR further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it, and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR which prevented the downstream interaction of Beclin1-PI3K-CIII, further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the aalpha;1 subunit was required for autophagy induction, since in aalpha;1 deficient cells neither AMPK nor autophagy were activated and HCMV was not inhibited by digitoxin. Summarized, induction of a novel pathway aalpha;1-AMPK-ULK1 induces autophagy as host-directed strategy for HCMV inhibition.

    IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally-infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs prompted evaluation of host-directed therapeutics. We report on a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the aalpha;1 subunit of the Na+, K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition since in aalpha;1- or ATG5-deficient cells virus was not inhibited. Furthermore, the AMPK activator, AICAR, antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. Summarized, autophagy may provide a strategy to harness HCMV replication.

  • Activities of Thrombin and Factor Xa are essential for replication of Hepatitis E virus and are possibly implicated in the ORF1 polyprotein processing [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a non-structural polyprotein of 1693 amino acids. It is not clear whether the ORF1 polyprotein is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave varied results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bear conserved cleavage sites of thrombin, and factor Xa. Using reverse genetics approach, we demonstrated that HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated in-vitro processing where we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of liver cell line with a serine protease inhibitor as well as siRNA knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in the liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.

    Importance: Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. High mortality rate of about 30% is observed in HEV infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to variable study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine protease thrombin and factor Xa that are conserved amongst HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study would shed light on the presence and roles of clotting factors with respect to virus replication in the cells.

  • The Amino-terminus of HSV-1 Glycoprotein K (gK) is Required for gB Binding to Akt, Release of Intracellular Calcium and Fusion of the Viral Envelope with Plasma Membranes [PublishAheadOfPrint]

  • Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino terminal 38 amino acids of gK prevents virus infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 (phospho S473) confirmed that HSV-1(McKrae) gB interacted with Akt-1 (phospo S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced release of intracellular calcium. In contrast, gB specified by HSV-1(McKrae) gK31-68 lacking the amino terminal 38 amino acids of gK failed to interact with Akt-1 (phospho S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae, but not gK31-68 in SK-N-SH cells. Importantly, entry of gK31-68, but not McKrae was significantly inhibited in SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate, indicating that McKrae gK31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate fusion of the viral envelope with cellular plasma membranes.

    IMPORTANCE HSV-1 glycoprotein B (gB) functions in fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino-terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.

  • TIP60 complex inhibits HBV transcription [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) is a global major health problem with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1 and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV cccDNA. Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents.

    IMPORTANCE Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.

  • A Cyclin-binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation [PublishAheadOfPrint]

  • Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, aa. 451-453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed as RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2, but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.

    IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in non-dividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.

  • Dengue virus selectively annexes endoplasmic reticulum-associated translation machinery as a strategy for co-opting host cell protein synthesis [PublishAheadOfPrint]

  • A primary question in Dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here we combined cell fractionation, ribosome profiling, and RNA-seq to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV (+) and (-) strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translational capacity was subverted from host transcripts to DENV (+) strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV (+) strand RNA and host mRNAs indicated that DENV (+) strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis-encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication, while minimizing the activation of anti-viral and proteostatic stress response pathways.


    DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle and thus targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.

  • Determinants in the IgV Domain of human HAVCR1 (TIM-1) are required to enhance Hepatitis C Virus Entry [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. The human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucin-like region, and was abrogated by a N94A mutation in the IgV phospholipid-binding pocket; indicating a direct interaction of the HAVCR1 IgV with HCV virions. However, knock out of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV although replacement of the mouse IgV domain with the human HAVCR1 IgV restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment leading to direct interactions with specific receptors, such as CD81.

    IMPORTANCE Hepatitis C virus (HCV) enters cells through a multi-faceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking, and silencing or knock out of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

  • Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation [PublishAheadOfPrint]

  • In the process of generating HSV-1 mutants in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine to proline (residue 393) and arginine to histidine (residue 950) substitutions in ICP6. To determine whether alteration(s) of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild type protein. Moreover, we noticed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.


    HSV-1 is a major human pathogen that infects ~80% of the human population and can be life threatening if neonates or immunocompromised individuals are infected. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuro-attenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and anti-apoptotic effect. Thus, we have determined that alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

  • HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs [PublishAheadOfPrint]

  • HIV-1 downregulates HLA-A and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing NK cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs, and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3-6.1-fold), which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to in vitro NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a common downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B and HLA-C exist within and between individuals, which can have functional consequences on immune recognition.

    IMPORTANCE Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in Sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome are often not used in experimental systems, due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of human leukocyte antigen (HLA) class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses, and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.

  • HIV-1 specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galcer Blocking and Phagocytosis [PublishAheadOfPrint]

  • Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against HIV-1 acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and their capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope specific IgA monoclonal antibodies by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, non-recombinant IgA monoclonal antibodies (mAbs). HG129 and HG130 mAbs mediated phagocytosis by monocytes and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g. multimeric, monomeric) in the systemic circulation and mucosal compartments.


    Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of anti-viral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and non-neutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the functional properties of HIV-1 specific IgG, more studies are needed on the functional attributes of HIV-1 specific IgA, specifically for vaccine-elicited IgA. Characterization of the functional properties of HIV-1 Env specific IgA monoclonal antibodies from human vaccine clinical trials are critical toward understanding the capacity of the host immune response to block HIV-1 acquisition.

  • ERK is a Critical Regulator of JC Polyomavirus Infection [PublishAheadOfPrint]

  • The human JC polyomavirus (JCPyV) infects the majority of the population worldwide and presents as an asymptomatic, persistent infection in the kidney. In individuals who are immunocompromised, JCPyV can become reactivated and cause a lytic infection in the central nervous system resulting in the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection is initiated by interactions between the capsid protein viral protein 1 (VP1) and the aalpha;2,6-linked sialic acid on LSTc, while JCPyV internalization is facilitated by 5-hydroxytryptamine 2 receptors (5-HT2Rs). The mechanisms by which the serotonin receptors mediate virus entry and the signaling cascades required to drive viral infection remain poorly understood. JCPyV was previously shown to induce phosphorylation of extracellular signal-regulated kinase (ERK), a downstream target of the mitogen-activated protein kinase (MAPK) pathway, upon virus entry. However, it remained unclear whether ERK activation was required for JCPyV infection. Both ERK-specific siRNA and ERK inhibitor treatments resulted in significantly diminished JCPyV infection in both kidney and glial cells, yet had no effect on the infectivity of polyomavirus simian virus 40 (SV40). Experiments characterizing the role of ERK during steps in the viral life cycle indicate that ERK activation is required for viral transcription as demonstrated by a significant reduction in production of the early viral gene large T-antigen (TAg), a key viral protein associated with the initiation of viral transcription and viral replication. These findings delineate the role of the MAPK-ERK signaling pathway in JCPyV infection, elucidating how the virus reprograms the host cell to promote viral pathogenesis.


    Viral infection is dependent upon host cell factors including the activation of cellular signaling pathways. These interactions between viruses and host cells are necessary for infection and play an important role in viral disease outcomes. The focus of this study was to determine how the human JC polyomavirus (JCPyV), a virus that resides in the kidney of the majority of the population and can cause the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brain of immunosuppressed individuals, usurps a cellular signaling pathway to promote its own infectious life cycle. We demonstrated that the activation of extracellular signal-regulated kinase (ERK), a component of the mitogen-activated protein kinase (MAPK) pathway, promotes JCPyV transcription, which is required for viral infection. Our findings demonstrate that the MAPK-ERK signaling pathway is a key determinant of JCPyV infection, elucidating new information regarding the signal reprogramming of host cells by a pathogenic virus.

  • Human Papillomavirus 16 infection induces VAP-dependent endosomal tubulation [PublishAheadOfPrint]

  • Human Papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans-Golgi network and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells we see that the defect in virus trafficking occurs post-capsid disassembly, but prior to localisation at the trans-Golgi network, with the incoming virion transduced DNA accumulating in Vps29/TGN46 positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations to endocytic transport pathways, some of which are VAP-dependent and required for endosome-to-Golgi transport of the incoming viral L2/DNA complex.

    Significance. Human Papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans-Golgi network. Indeed, loss of VAP blocks HPV infectious entry at a step post-capsid uncoating, but prior to localisation at the trans- Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry.

  • Arabidopsis RNA Polymerase V Mediates Enhanced Compaction and Silencing of Geminivirus and Transposon Chromatin During Host Recovery from Infection [PublishAheadOfPrint]

  • Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for Arabidopsis thaliana to recover from infection with Beet curly top virus lacking a suppressor protein that inhibits methylation (BCTV L2-). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) to confirm that ggt;90% of BCTV L2- chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V, and by extension the Pol IV-RdDM pathway. BCTV L2- was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons Ta3 and Athila6A. Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely especially important for natural recovery from wild type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues.

    IMPORTANCE In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission, and thus allows normal floral development. Studies described here demonstrate that Pol V-dependent enhanced viral DNA and histone methylation observed during recovery results in increased chromatin compaction and suppressed gene expression. In addition, we show that TE associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue.

  • Cell-to-cell measles virus spread between human neurons dependent on the hemagglutinin and the hyperfusogenic fusion protein [PublishAheadOfPrint]

  • Measles virus (MV) usually causes acute infection, but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule (SLAM) and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We here show that a hyperfusogenic mutant MV (IC323-F(T461I)-EGFP), but not the wild-type MV, spreads in differentiated NT2 cells, a widely-used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting the cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by the fusion inhibitor peptide as well as by some but not all of anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation, and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin and the putative neuronal receptor for MV.

    IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by the fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

  • Class 1-selective histone deacetylase inhibitors enhance HIV latency reversal while preserving the activity of HDAC isoforms necessary for maximal HIV gene expression [PublishAheadOfPrint]

  • Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of PKC agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of pro-viral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat plus bryostatin-1 induced viral outgrowth, whereas bryostatin-1- combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-B and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate limitations of current approaches and provide a path towards more effective strategies to eliminate the HIV reservoir.

    IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly-acting agents. Additionally, we provide evidence that broadly-acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of pro-viral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.

  • Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors [PublishAheadOfPrint]

  • Human and chimpanzee adenovirus vectors are being developed to circumvent pre-existing antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes two months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development.

    IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development.

  • A role for myosin Va in human cytomegalovirus nuclear egress [PublishAheadOfPrint]

  • Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the bbeta;-herpesvirus, human cytomegalovirus, and is important for nuclear egress and capsid localization away from replication compartment-like inclusions towards the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. Here, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and co-immunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions towards the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress.

    IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of aalpha;-herpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here, we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin, and that antagonism of myosin Va impairs capsid localization towards the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved.

  • Cleavage of the C-terminal fragment of reovirus {mu}1 is required for optimal infectivity [PublishAheadOfPrint]

  • The mammalian orthoreovirus (reovirus) outer capsid, which is composed of 200 mmu;1/3 heterohexamers and a maximum of 12 1 trimers, contains all of the proteins that are necessary for attaching to and entering host cells. Following attachment, reovirus is internalized by receptor-mediated endocytosis and acid-dependent cathepsin proteases degrade the 3 protein. This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the mmu;1 membrane penetration protein is exposed. ISVPs undergo a second structural rearrangement to deposit the genome-containing core into the host cytoplasm. The conformationally altered particle is called ISVP*. ISVP-to-ISVP* conversion culminates in the release of mmu;1 N- and C-terminal fragments, mmu;1N and , respectively. Released mmu;1N is thought to facilitate core delivery by generating size-selective pores within the endosomal membrane, whereas the precise role of , particularly in the context of viral entry, is undefined. In this report, we characterize a recombinant reovirus that fails to cleave from mmu;1 in vitro. cleavage, which is not required for ISVP-to-ISVP* conversion, enhances the disruption of liposomal membranes and facilitates the recruitment of ISVP*s to the site of pore formation. Moreover, the cleavage-deficient strain initiates infection of host cells less efficiently than the parental strain. These results indicate that mmu;1N and contribute to reovirus pore forming activity.

    IMPORTANCE Host membranes represent a physical barrier that prevents infection. To overcome this barrier, viruses utilize diverse strategies, such as membrane fusion or membrane disruption, to access internal components of the cell. These strategies are characterized by discrete protein-protein and protein-lipid interactions. The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of well-defined conformational changes, which conclude with pore formation and delivery of the viral genetic material. In this report, we characterize the role of the small, reovirus-derived peptide in pore formation. cleavage from the outer capsid enhances membrane disruption and facilitates the recruitment of virions to membrane associated pores. Moreover, cleavage promotes the initiation of infection. Together, these results reveal an additional component of the reovirus pore forming apparatus and highlight a strategy for penetrating host membranes.

  • Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against Zika Virus Infection and Testis Damage [PublishAheadOfPrint]

  • The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitos as seen for classical arboviruses. We and others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available, therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low pre-existing immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week post-vaccination and the elicited antisera could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mice sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against the ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control.

    IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barreeacute; syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid pre-existing anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M/E glycoproteins was used for ZIKV vaccine development. Impressively, AdC7-M/E exhibited exceptional performance as a ZIKV vaccine: 1) protective efficacy by a single vaccination; 2) rapid development of a robust humoral response; 3) durable immune responses; 4) robust T cell responses; and 5) sterilizing immunity achieved by a single vaccination. These advantages of AdC7-M/E strongly support its potential application as a promising ZIKV vaccine in the clinic.

  • Host Tumor Suppressor p18INK4c functions as a potent cell-intrinsic inhibitor of {gamma}HV68 reactivation and pathogenesis. [PublishAheadOfPrint]

  • Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (HV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor, p18INK4c (p18). Whether p18 plays a cell-intrinsic or nndash;extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and in infected cell reservoirs. While the cycKO virus is capable of reactivation in p18 deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict HV68 pathogenesis even in p18 sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells, and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.

    IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection, which is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.

  • Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate With The Viral Envelope as a Type I Integral Membrane Protein [PublishAheadOfPrint]

  • The glycosylated Gag protein (gPr80) of murine leukemia viruses (MLVs) has been shown to exhibit multiple roles in facilitating retrovirus release, infection and resistance to host-encoded retroviral restriction factors such as APOBEC3, SERINC3 and SERINC5. One way gPr80 helps MLVs escape host innate immune restriction is by increasing capsid stability, a feature that protects viral replication intermediates from being detected by cytosolic DNA sensors. gPr80 also increases the resistance of MLVs against deamination and restriction by mouse APOBEC3 (mA3). How the gPr80 accessory protein, with its three N-linked glycosylation sites, contributes to these resistance mechanisms is still not fully understood. Here we have further characterized the function of gPr80 and, more specifically, revealed that the asparagines targeted for glycosylation in gPr80 also contribute to capsid stability through their parallel involvement in the Pr65 Gag structural polyprotein. In fact, we demonstrate that sensitivity to deamination by mA3 and human A3 proteins is directly linked to capsid stability. We also show that full-length gPr80 is detected in purified viruses. However, our results suggest that gPr80 is inserted in the NexoCcyto orientation of a type I integral membrane protein. Additionally, our experiments have revealed the existence of a large population of Env-deficient virus-like particles (VLPs) harbouring gPr80 inserted in the opposite (NcytoCexo) polarity which is typical of type II integral membrane proteins. Overall this study provides new insight into the complex nature of the MLV gPr80 accessory protein.

    IMPORTANCE Viruses have evolved numerous strategies to infect, spread and persist in their host. Here we analyze the details of how the MLV-encoded glycosylated Gag (gPr80) protein protects the virus from being restricted by host innate immune defenses. gPr80 is a variant of the structural Pr65 Gag protein with an 88 amino acid extended leader sequence that directs the protein for translation and glycosylation in the endoplasmic reticulum. This study dissects the specific contributions of gPr80 glycans and capsid stability in helping the virus infect, spread and counteract the effects of the host intrinsic restriction factor APOBEC3. Overall this study provides further insight into the elusive role of the gPr80 protein.

  • Genomic and Biochemical Characterization of Acinetobacter Podophage Petty Reveals a Novel Lysis Mechanism and Tail-Associated Depolymerase Activity [PublishAheadOfPrint]

  • The increased prevalence of drug-resistant, nosocomial Acinetobacter infections, particularly from pathogenic members of the Acinetobacter calcoaceticus-baumannii complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterize phage Petty, a novel podophage that infects multidrug-resistant Acinetobacter nosocomialis and Acinetobacter baumannii. Genome analysis reveals that phage Petty is a 40,431bp KMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single subunit endolysin, but lacks canonical spanins to disrupt the outer membrane. Analysis of other KMV-like genomes revealed that spanin-less lysis cassettes are a feature of phages infecting Acinetobacter within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). Gene 39, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4 kDa protein gp39 was cloned and expressed, and its activity against Acinetobacter EPS in solution was determined. The enzyme degraded purified EPS from its host strain A. nosocomialis AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant Acinetobacter strains.

    Importance Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for Acinetobacter infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data needs to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this study, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported able to infect both A. baumannii and A. nosocomialis. The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome, and confirm that, unlike the rounding of lysogens lacking functional spanin genes, A. nosocomialis cells infected with phage Petty lyse by bursting. This suggests phages like Petty employ a different mechanism to disrupt the outer membrane of Acinetobacter hosts during lysis.

  • Evasion of Cytosolic DNA-Stimulated Innate Immune Responses by HSV-1 [PublishAheadOfPrint]

  • Recognition of virus-derived nucleic acids by host pattern recognition receptors (PRRs) is crucial for early defense against viral infections. Recent studies revealed that PRRs also include several newly identified DNA sensors, most of which could activate the downstream adaptor stimulator of interferon genes (STING) and lead to the production of host antiviral factors. Herpes simplex virus type 1 (HSV-1) is extremely successful to establish an effective infection due to its capacity to counteract host innate antiviral responses. In the present review, I summarized the most recent findings on the molecular mechanisms utilized by HSV-1 to target different steps of the cellular DNA sensor-mediated antiviral signal pathway.

  • The simultaneous insertion of two ligands in gD for the cultivation of oncolytic HSVs in non-cancer cells and the retargeting to cancer receptors [PublishAheadOfPrint]

  • Insertion of a single chain antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Clinical grade virus production can not rely on cancer cells. Recently, we developed a double retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the non-cancer producer Vero-GCN4R cell line, and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more advantageous detargeting strategies were devised, so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of aa 35-39, 214-223, or 219-223, and replacement of the deleted sequences with one of the two ligands. The latter two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best performing recombinants carried a gD deletion as small as 5 amino acids, and grew to titers similar to those exhibited by the singly retargeted R-LM113, and by the non-retargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effective in vitro and in vivo.


    There is increasing interest in oncolytic viruses, following FDA and EMA approval of the oncolytic HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in non-cancer cells. Here, we describe a double retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative, one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally-disadvantageous detargeting modifications. The current and accompanying studies teach how to best achieve the clinical-grade cultivation of retargeted oncolytics.

  • Dual ligand insertion in gB and in gD of oncolytic HSVs for the retargeting to a producer Vero cell line and to cancer cells. [PublishAheadOfPrint]

  • Oncolytic viruses gain cancer specificity in several ways. Like the majority of viruses, they grow better in cancer cells which are defective in mounting the host response to viruses. Often they are attenuated by deletion or mutation of virulence genes which counteract the host response, or are naturally occurring oncolytic mutants. In contrast, retargeted viruses are not attenuated or deleted; their cancer-specificity rests on a modified, specific tropism for cancer receptors. For herpes simplex virus (HSV)-based oncolytics, the detargeting-retargeting strategies employed so far were based on genetic modifications of gD. Recently, we showed that even gH or gB can serve as retargeting tools. To enable the growth of retargeted HSVs in cells that can be used for clinical grade virus production, a double retargeting strategy has been developed. Here we show that several sites in the N-terminus of gB are suitable to harbour the 20 aa long GCN4 peptide, which readdresses HSV tropism to Vero cells expressing the artificial GCN4 receptor, and thus enables virus cultivation in the producer non-cancer Vero-GCN4R cell line. The gB modifications can be combined with a minimal detargeting modification in gD, consisting in the deletion of two residues, aa 30 and 38, and replacement of aa 38 with the scFv to HER2, for retargeting to the cancer receptor. The panel of recombinants was analysed comparatively in terms of virus growth, cell-to-cell spread, cytotoxicity, in vivo anti-tumor efficacy to define the best double retargeting strategy.


    There is increasing interest in oncolytic viruses, following FDA and EMA approval of HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly checkpoint inhibitors. A strategy to gain cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. Cultivation of fully retargeted viruses is challenging, since they require cells that express the cancer receptor. We devised a strategy for their cultivation in producer non-cancer Vero cell derivative. Here, we developed a double retargeting strategy, based on insertion of one ligand in gB for retargeting to Vero cell derivative, and of anti-HER2 ligand in gD for cancer retargeting. These modifications were combined with a minimally-destructive detargeting strategy. Current and accompanying study teach the clinical-grade cultivation of retargeted oncolytic HSVs, and promote their translation to the clinic.

  • Actin-dependent non-lytic rotavirus exit and infectious virus morphogenetic pathway in non-polarized cells [PublishAheadOfPrint]

  • During the late stages of rotavirus morphogenesis the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from non-polarized MA104 cells at early times of infection when there is still no evidence of cell lysis. These findings are in contrast with the traditional assumption that rotavirus is released from non-polarized cells by a non-specific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular media by density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein.

    IMPORTANCE Rotavirus is a major etiological agent of infantile acute severe diarrhea. It is a non-enveloped virus formed by three concentric layers of protein. The early stages of rotavirus replication, including cell attachment and entry, synthesis and translation of viral mRNAs, replication of the genomic dsRNA, and the assembly of double-layered viral particles, have been widely studied. However, the mechanism involved in the later stages of infection, i.e, viral particle maturation and cell exit, have been less characterized. It has been historically assumed that rotavirus exits non-polarized cells following cell lysis. In this work, we show that the virus exits cells by a non-lytic, actin-dependent mechanism and, most importantly, we describe that VP4, the spike protein of the virus, is present on the cell surface and is incorporated into mature, infectious virus, indicating a novel pathway for the assembly of this protein.

  • Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments [PublishAheadOfPrint]

  • The neutralizing antibody (nAb) response against the influenza virus's hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency on inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor-binding pocket at HA's distal end, while FI6v3 binds primarily to the HA2 fusion subunit towards the base of the stalk. Surprisingly, HC19 inhibited HA's ability to induce lipid mixing by preventing structural rearrangement of HA under fusion activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding, but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to crosslink separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that inter-spike crosslinking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared down Fab-soluble HA structures.


    The influenza virus's hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of Ab-mediated neutralization largely relies on high resolution characterization of antigen binding fragments (Fab) in complex with soluble, isolated antigen constructs by cryo-EM single particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well-characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occurs by multiple coexisting mechanisms and is largely dependent on the specific epitope that is targeted and is highly dependent on the bivalent nature of IgG molecules.

  • Asian elephant T cell responses to Elephant Endotheliotropic Herpesvirus (EEHV) [PublishAheadOfPrint]

  • Elephant Endotheliotropic Herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, an endangered species. One hypothesis to explain this vulnerability of some juvenile elephants is that they fail to mount an effective T cell response to the virus. To our knowledge, there have been no studies of Asian elephant T cell responses to EEHV. To address this deficiency, we validated the IFN- ELISpot assay for tracking antigen-directed T cell activity by monitoring rabies-specific responses in vaccinated elephants. Additionally, we generated monoclonal antibodies to Asian elephant CD4 and CD8 to facilitate phenotypic T cell profiling. Using these tools, we screened healthy elephants with a prior history of EEHV infection for reactivity against 9 EEHV proteins whose counterparts in other herpesviruses are known to induce T cell responses in their natural hosts. We identified glycoprotein B (gB) and the putative regulatory protein E40 as the most immunogenic T cell targets (IFN- responses in 5 of 7 elephants) followed by the major capsid protein (MCP) (IFN- responses in 3 of 7 elephants). We also observed that IFN- responses were largely from CD4+ T cells. We detected no activity against the predicted major immediate early (E44) and large tegument (E34) proteins- both immunodominant T cell targets in humans latently infected with cytomegalovirus. These studies have identified EEHV-specific T cells in Asian elephants for the first time, lending insight into the T cell priming that might be required to protect against EEHV disease and will guide the design of effective vaccine strategies.

    IMPORTANCE Endangered Asian elephants are facing many threats, including lethal hemorrhagic disease from elephant endotheliotropic herpesvirus (EEHV). EEHV usually establishes chronic, benign infections in mature Asian elephants but can be lethal to juvenile elephants in captivity and the wild. It is the leading cause of death in captive Asian elephants in North America and Europe. Despite availability of sensitive tests and protocols for treating EEHV-associated illness, these measures are not always effective. The best line of defense would be a preventative vaccine. We interrogated normal healthy elephants previously infected with EEHV for T cell responses to 9 EEHV proteins predicted to induce cellular immune responses. Three proteins elicited IFN- responses, suggesting their potential usefulness as vaccine candidates. Our work is the first to describe T cell responses to a member of the proposed fourth subfamily of mammalian herpesviruses, the Deltaherpesvirinae, within a host species in the clade Afrotheria. An EEHV vaccine would greatly contribute to the healthcare of Asian and African elephants that are also susceptible to this disease.

  • Cryo-EM Structure of Seneca Valley Virus Procapsid [PublishAheadOfPrint]

  • Seneca Valley Virus, like some other members of the picornaviridae, forms naturally occurring empty capsids, known as procapsids. The procapsid has the same antigenicity as the full virion, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumour cells, and is being explored for use as a therapeutic agent in the treatment of small cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid-stable.


    Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley Virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.

  • RSAD2 and AIM2 modulate CV-A16 and EV-A71 replication in neuronal cells in different ways that may be associated with their 5' non-translated regions [PublishAheadOfPrint]

  • Coxsackievirus A16 (CV-A16) and Enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot and mouth disease but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and qRT-PCR in CV-A16 infected neuroblastoma cells (SK-N-SH) which showed that the radical s-adenosyl methionine domain containing 2 (RSAD2) was the highest up-regulated gene in the anti-microbial pathway. Increased RSAD2 expression was correlated with reduced viral replication while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2) which is associated with pyroptosis cell death was upregulated in EV-A71 infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5' non-translated regions (5' NTR) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5' NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5' NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16 associated neurovirulence in HFMD outbreaks compared to EV-A71 infection.

    IMPORTANCE Although Coxsackievirus A16 (CV-A16) and Enterovirus A17 (EV-A71) both cause hand, foot and mouth disease, EV-A71 has emerged as a leading cause of non-polio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact on viral neuropathogenesis. We further showed that viral 5' NTRs may play significant roles in eliciting different host response mechanisms.

  • Emerging Alphaviruses are Sensitive to Cellular States Induced by a Novel Small Molecule Agonist of the STING Pathway [PublishAheadOfPrint]

  • The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this we employed a high-throughput screen to identify small cell-permeable molecules capable of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(Methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss of function assays we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging Alphavirus types including Chikungunya, Ross River, Venezuelan Equine Encephalitis, Mayaro, and O'nyong'nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as crucial to cellular resistance to Alphavirus infection.

    IMPORTANCE: Due to the increase in emerging arthropod-borne viruses like Chikungunya that lack FDA-approved therapeutics and vaccines, it is important to better understand signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, and supports its value in understanding the immune response and viral pathogenesis required to establish host infection. We also show C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

  • The ATM and Rad3-related (ATR) Protein Kinase Pathway is Activated by Herpes Simplex Virus 1 (HSV-1) and Required for Efficient Viral Replication [PublishAheadOfPrint]

  • The ATM and Rad3-related (ATR) protein kinase, and its downstream effector Chk1, are key sensors and organizers of the DNA damage response (DDR) to a variety of insults. Previous studies of Herpes Simplex Virus 1 (HSV-1) showed no evidence for activation of the ATR pathway. Here we demonstrate that both Chk1 and ATR were phosphorylated by 3 h.p.i.. Activation of ATR and Chk1 was observed using 4 different HSV-1 strains in multiple cell types, while a specific ATR inhibitor blocked activation. Mechanistic studies point to early viral gene expression as a key trigger for ATR activation. Both pATR and pChk1 localized to the nucleus within viral replication centers, or associated with their periphery, by 3 h.p.i.. Significant levels of pATR and pChk1 were also detected in the cytoplasm where they co-localized with ICP4 and ICP0. Proximity ligation assays confirmed that pATR and pChk1 were closely and specifically associated with ICP4 and ICP0 in both the nucleus and cytoplasm by 3 h.p.i., but not with ICP8 or ICP27, presumably in a multiprotein complex. Chemically distinct ATR and Chk1 inhibitors blocked HSV-1 replication and infectious virion production, while inhibitors of ATM, Chk2, and DNA-PK did not. Together our data show that HSV-1 activates the ATR pathway at early stages of infection, and that ATR and Chk1 kinase activities play important roles in HSV-1 replication fitness. These findings indicate that the ATR pathway may provide insight for therapeutic approaches.

    IMPORTANCE Viruses have evolved complex associations with cellular DNA damage response (DDR) pathways, which sense troublesome DNA structures formed during infection. The first evidence for activation of the ATR pathway by HSV-1 is presented. ATR is activated, and its downstream target Chk1 is robustly phosphorylated, during early stages of infection. Both activated proteins are found in the nucleus associated with viral replication compartments, and in the cytoplasm associated with viral proteins. We also demonstrate that both ATR and Chk1 kinase activities are important for viral replication. The findings suggest that HSV-1 activates ATR and Chk1 during early stages of infection, and utilizes the enzymes to promote its own replication. The observation may be exploitable for antiviral approaches.

  • Efficient translation of EBV DNA polymerase contributes to the enhanced lytic replication phenotype of M81 EBV. [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is linked to development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV isolated from a Chinese patient with nasopharyngeal carcinoma (NPC) demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons have previously been performed between M81 and B95-8 EBV in order to determine if the hyper-lytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA binding protein expressed during early lytic replication that is essential for virus production. We compared the function of M81 and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, expression of the EBV DNA polymerase protein was much higher in M81 compared to B95-8 infected cells. The relative deficiency in expression of B95-8 DNA polymerase was related to the B95-8 genome deletion which truncates the BALF5 3' UTR. Similarly, insertion of bacmid DNA in the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that enhanced lytic replication and virus production of M81 compared to B95-8 is partly due to robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81.

    IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population but the incidence of EBV associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphism from patient isolates of EBV, gene expression phenotype and disease is important and may help in developing antiviral therapy. This study examines potential causes for the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3' UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis.

  • Antagonism of the protein kinase R pathway in human cells by rhesus cytomegalovirus [PublishAheadOfPrint]

  • While cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a singular host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2aalpha;, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2-3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells.

    IMPORTANCE Rhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is only capable of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor.

  • The Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING [PublishAheadOfPrint]

  • The innate immune response plays a pivotal role during human cytomegalovirus (HCMV) primary infection. Indeed, HCMV infection of primary fibroblasts rapidly triggers strong induction of type I interferons (IFN-I) accompanied by proinflammatory cytokine release. Here, we show that primary human foreskin fibroblasts (HFFs) infected with a mutant HCMV TB40/E strain unable to express UL83-encoded pp65 (v65Stop) produce significantly higher IFN-bbeta; levels than HFFs infected with the wild-type TB40/E strain or the pp65 revertant (v65Rev), suggesting that the tegument protein pp65 may dampen IFN-bbeta; production. To clarify the mechanisms through which pp65 inhibits IFN-bbeta; production, we analyzed the activation of the cGAS/STING/IRF3 axis in HFFs infected with either wild-type, v65Rev or the pp65-deficient mutant v65Stop. We found that pp65 selectively binds to cGAS and prevents its interaction with STING, thus inactivating the signaling pathway through the cGAS/STING/IRF3 axis. Consistently, addition of exogenous cGAMP to v65Rev infected cells triggered the production of IFN-bbeta; levels similar to those observed with v65Stop infected cells confirming that pp65 inactivation of IFN-bbeta; production occurs at the cGAS level. Notably, within the first 24 hours of HCMV infection, STING undergoes proteasome degradation independent of the presence or absence of pp65. Collectively, our data provide mechanistic insights into the interplay between HCMV pp65 and cGAS, leading to subsequent immune evasion by this prominent DNA virus.

    IMPORTANCE Primary human foreskin fibroblasts (HFFs) produce type I IFN (IFN-I) when infected with HCMV. However, we observed significantly higher IFN-bbeta; levels when HFFs were infected with HCMV unable to express UL83-encoded pp65 (v65Stop), suggesting that pp65 (pUL83) may constitute a viral evasion factor. This study demonstrates that HCMV tegument protein pp65 inhibits IFN-bbeta; production by binding and inactivating cGAS early during infection. In addition, this inhibitory activity specifically targets cGAS since it can be bypassed via the addition of exogenous cGAMP, even in presence of pp65. Notably, STING proteasome-mediated degradation was observed in both the presence and absence of pp65. Collectively, our data underscore the important role of tegument protein pp65 as a critical molecular hub in HCMV's evasion strategy to the innate immune response.

  • Naturally Occurring Frame-Shift Mutations in the tvb Receptor Gene Are Responsible for Decreased Susceptibility of Chicken to Infection with Avian Leukosis Virus Subgroups B, D, and E [PublishAheadOfPrint]

  • The group of highly related avian leucosis viruses (ALVs) in chickens thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells. Host exposed to ALVs might be under selective pressure to develop resistance to ALVs infection. Indeed, the resistant alleles have previously been identified in all four receptor loci in chicken. The tvb gene encodes a receptor, which determines the susceptibility of host cells to the subgroups B, D, and E ALV. We herein describe the identification of two novel alleles of tvb receptor gene, which possess independent insertions each within the exon 4. The insertions resulted in frame-shift mutations reveal a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the production of truncated Tvb protein. As a result, we observed that the frame-shift mutations in the tvb gene significantly lower the binding affinity of the truncated Tvb receptors for the ALV-B, ALV-D and ALV-E envelope glycoproteins, and significantly reduce susceptibility to infection by ALV-B, ALV-D and ALV-E in vitro and in vivo. Taken together, these findings are suggestive for frame-shift mutation can be a molecular mechanism of reduction of susceptibility to ALV and enhance our understanding of virus-host coevolution.

    IMPORTANCE Avian leukosis virus (ALV) once caused devastated economic loss to the U.S. poultry industry prior the current eradication schemes in place, and continues causing severe calamity to the poultry industry in China and Southeast Asia, where deployment of a complete eradication scheme remains a challenge. The tvb gene encodes the cellular receptor necessary for the subgroup B, D, and E ALV infection. Two tvb allelic variants resulted from frame-shift mutations have been identified in this study, which have been elucidated with significantly reduced functionality in mediating the subgroups B, D, and E ALV infection. Unlike the control of herpesvirus-induced diseases by vaccination, the control of avian leukosis in chickens has been totally relying on virus eradication measures and host genetic resistance. This finding enriches the allelic pool of tvb gene, and expands the potentiality for genetic improvement of ALV resistance in varied chicken populations by selection.

  • The human papillomavirus E6 oncoprotein targets USP15 and TRIM25 to suppress RIG-I-mediated innate immune signaling [PublishAheadOfPrint]

  • Retinoic acid-inducible gene-I (RIG-I) is a key pattern-recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25 stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-bbeta;, chemokines and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene-targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains, and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.

    IMPORTANCE Persistent infection and tumorigenesis by human papillomaviruses (HPVs) are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.

  • GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors [PublishAheadOfPrint]

  • Although it has been shown that some of the mannose-binding lectins (MBLs) exhibit significant activity against HIV infection, little is known about whether N-acetyl-D-galactosamine (GalNAc)-binding lectins have the ability to inhibit HIV infection. We here demonstrate that a soybean-derived lectin (SBL) with GalNAc-binding affinity could potently suppress HIV infection of macrophages in a dose-depend fashion. Unlike the MBLs that block HIV only through binding to the glycosylated envelope proteins (gp120, gp41) of the virus, SBL inhibited HIV at multiple steps of the virus infection/replication cycle. SBL could activate IFN-bbeta;-STAT signaling pathway, resulting in the upregulation of a number of antiviral ISGs in macrophages. In addition, SBL treatment of macrophages induced the production of C-C chemokines, which bind to HIV entry coreceptor CCR5. Deglycosylation of cell surface galactosyl moieties or pre-saturation of GalNAc-binding capacity could compromise SBL-mediated induction of the antiviral factors. Furthermore, SBL exerted its anti-HIV activity in the low nanomolar range with no mitogenic effect on CD4+ T cells, a major advantage in the development of SBL as a potential anti-HIV agents as compare with MBLs. These data indicate a necessity to further investigate SBL as an alternative and cost-effective anti-HIV natural product.

    IMPORTANCE Mannose-binding lectins (MBLs) can block the attachment of HIV to target cells and are suggested as anti-HIV microbicides. However, the mitogenic effect of MBLs on CD4+ T cells limits this potential in clinical settings. Lectins with galactose (Gal) or N-acetyl-D-galactosamine (GalNAc)-binding specificity are another important category of carbohydrate-binding proteins (CBPs). As compared to high-mannose N-linked glycans, GalNAc-type glycans present much less in HIV gp120 or gp41 glycosylation. We here demonstrated that GalNAc-specific soybean lectin (SBL) triggers an antiviral signaling via recognition of the cell surface galactosyl group of macrophages, which resulted in the suppression of HIV at multiple steps. More importantly, SBL has no mitogenic effect on the activation of CD4+ T cells, a major advantage in the development of Gal/GalNAc-specific lectins as naturopathic anti-HIV agents.

  • Identification of a conserved interface of HIV-1 and FIV Vifs with Cullin 5 [PublishAheadOfPrint]

  • The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC3, A3) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the APOBEC3 (A3) genes encode for the A3Z2s, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for interaction with Cullin are poorly understood. Here, we found that the expression of dominant-negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant-negative CUL2 had no influence on the degradation of A3. In co-immunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from position 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175mmdash; C161mmdash;C184mmdash;C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for CUL5 interaction. Mutating this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif/CUL5 interaction, residues 52LW53 in CUL5 were identified as mediating the binding to FIV Vif. By comparing our results to the HIV-1 Vif/CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vif is evolutionarily conserved indicating a strong structural constraint. However, the FIV Vif/CUL5 interaction is zinc-independent, which contrasts with the zinc-dependence of HIV-1 Vif.

    IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus (HIV)-1, replicates in its natural host in T-cells and macrophages that express antiviral restriction factors APOBEC3 (A3). To escape A3s, FIV and HIV induce degradation of these proteins by building ubiquitination ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc in contrast to HIV-1 Vif.

  • The Host Factor AUF1 p45 Supports Flavivirus Propagation by Triggering the RNA Switch Required for Viral Genome Cyclization [PublishAheadOfPrint]

  • In previous studies, we showed that the cellular RNA-binding protein AUF1 supports the replication process of the Flavivirus West Nile. Here, we demonstrate that the protein also enables effective proliferation of Dengue virus and Zika virus, indicating AUF1 is a general Flavivirus host factor. Further studies demonstrated that the AUF1 isoform p45 significantly stimulates the initiation of viral RNA replication and that the protein's RNA chaperone activity enhances the interactions of the viral 5' UAR- and 3' UAR genome cyclization sequences. Most interestingly, we observed that AUF1 p45 destabilizes not only the 3' -terminal stem-loop (3'SL) but also the 5' -terminal stem-loop B (SLB) of the viral genome. RNA structure analyses revealed that AUF1 p45 increases the accessibility of defined nucleotides within the 3'SL and the SLB, and, in this way, exposes both UAR cyclization elements. Conversely, AUF1 p45 does not modulate the fold of stem-loop A (SLA) at the immediate genomic 5' -end, which is proposed to function as a promoter of the viral RNA-dependent RNA polymerase (RdRp). These findings suggest that AUF1 p45, by destabilizing specific stem-loop structures within the 5' - and 3' -end of the flaviviral genome, assists genome cyclization and, concurrently, enables the RdRp to initiate RNA synthesis. Our study thus highlights the role of a cellular RNA-binding protein inducing a flaviviral RNA switch that is crucial for viral replication.

    IMPORTANCE The genus Flavivirus within the Flaviviridae family includes important human pathogens such as Dengue-, West Nile-, and Zika virus. The initiation of replication of the flaviviral RNA genome requires a transformation from a linear to a cyclized form. This involves considerable structural reorganization of several RNA motifs at the genomic 5' - and 3' -ends. Specifically, it needs a melting of stem-structures to expose complementary 5' - and 3' cyclization elements to enable their annealing during cyclization. Here, we show that a cellular RNA chaperone, AUF1 p45, which supports the replication of all three aforementioned Flaviviruses, specifically rearranges stem-structures at both ends of the viral genome and, in this way, permits 5' -3' -interactions of cyclization elements. Thus, AUF1 p45 triggers the RNA switch in the flaviviral genome that is crucial for viral replication. These findings represent an important example of how cellular (host) factors promote the propagation of RNA viruses.

  • Major histocompatibility complex class I (FLA-E*01801) molecular structure in domestic cats demonstrates species-specific characteristics in presenting viral antigen peptides [PublishAheadOfPrint]

  • Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag, 39-48; RMANVSTGR, RMA9 for short). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and in vitro refolding of the mutant FLA-E*01801 complex demonstrated that Glu63 and Trp167 in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine.


    Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp167 blocks the left side of pocket A, causing P1D to conflict with Glu63. We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We determined 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC I molecules present peptides.


  • Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, and an effective vaccine is not yet available. We previously generated an RSV live-attenuated vaccine (LAV) candidate "DB1" which was attenuated by a low-fusion subgroup B F protein (BAF) and codon-deoptimized non-structural protein genes. DB1 was immunogenic and protective in cotton rats, but lacked thermostability and stability of the pre-fusion conformation of F compared to strains with the line19 F gene. We hypothesized that incorporation of unique residues from the thermostable A2-line19F strain could thermostabilize DB1 and boost its immunogenicity. We therefore incorporated 4 unique line19F residues into the BAF protein of DB1 by site-directed mutagenesis and rescued the recombinant virus "DB1-QUAD." Compared to DB1, DB1-QUAD had improved thermostability at 4ddeg;C and higher levels of pre-fusion F as measured by ELISAs. DB1-QUAD was attenuated in normal human bronchial epithelial cells, in BALB/c mice, and in cotton rats, but grew to wild-type titers in Vero cells. In mice, DB1-QUAD was highly immunogenic and generated significantly higher neutralizing antibody titers to a panel of RSV A and B strains than did DB1. DB1-QUAD was also efficacious against wild-type RSV challenge in mice and cotton rats. Thus, incorporation of unique line19F residues into RSV LAV DB1 enhanced vaccine thermostability, incorporation of pre-fusion F, and immunogenicity and generated a promising vaccine candidate that merits further investigation.

    IMPORTANCE We boosted the thermostability and immunogenicity of an RSV live-attenuated vaccine candidate by incorporating 4 unique residues from the RSV line19F protein into the F protein of heterologous vaccine strain DB1. The resultant vaccine candidate "DB1-QUAD" was thermostable, attenuated in vivo, highly immunogenic, and protective against RSV challenge in mice and cotton rats.

  • Identification of residues controlling restriction versus enhancing activities of IFITM proteins on the entry of human coronaviruses [PublishAheadOfPrint]

  • Interferon-induced transmembrane proteins (IFITM) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV)-OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the role of structural motifs important for IFITM protein post-translational modifications, intracellular trafficking and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance the entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, substitution of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic the unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and 229E, but enhanced the entry of SARS-CoV and MERS-CoV. Second, substitution of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV, but promoted the entry of MERS-CoV. Third, deletion of carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most possibly through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.

    IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein mediated entry reveals key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved a way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

  • IL-10 modulation of virus clearance and disease in mice with alphaviral encephalomyelitis [PublishAheadOfPrint]

  • Alphaviruses are an important cause of mosquito-borne outbreaks of arthritis, rash and encephalomyelitis. Previous studies in mice with a virulent strain (NSV) of the alphavirus Sindbis virus (SINV) identified a role for Th17 cells and regulation by IL-10 in the pathogenesis of fatal encephalomyelitis (K.A. Kulcsar, V.K. Baxter, I.P. Greene and D.E. Griffin, Proc Natl Acad Sci USA 111:16053-16058, 2014). To determine the role of virus virulence in generation of immune responses, we analyzed the modulatory effects of IL-10 on disease severity, virus clearance and the CD4+ T cell response to infection with a recombinant strain of SINV of intermediate virulence (TE12). The absence of IL-10 during TE12 infection led to prolonged morbidity, more weight loss, higher mortality and slower viral clearance compared to wild-type mice. More severe disease and impaired virus clearance in IL-10-/- mice were associated with more Th1 cells; fewer Th2 cells, innate lymphoid type 2 cells, regulatory cells and B cells and delayed production of antiviral antibody in the central nervous system (CNS) without an effect on Th17 cells. Therefore, IL-10 deficiency led to more severe disease in TE12-infected mice by increasing Th1 cells and by hampering development of the local B cell responses necessary for rapid production of antiviral antibody and virus clearance from the CNS. In addition, the shift from Th17 to Th1 responses with decreased virus virulence indicates that the effects of IL-10 deficiency on immunopathologic responses in the CNS during alphavirus infection are influenced by virus strain.

    IMPORTANCE Alphaviruses cause mosquito-borne outbreaks of encephalomyelitis, but determinants of outcome are incompletely understood. We analyzed the effects of an anti-inflammatory cytokine IL-10 on disease severity and virus clearance after infection with an alphavirus strain of intermediate virulence. The absence of IL-10 led to prolonged illness, more weight loss, more death and slower viral clearance compared to mice that produced IL-10. IL-10 influenced development of disease-causing T cells and entry into the brain of B cells producing antiviral antibody. The Th1 pathogenic cell subtype that developed in IL-10-deficient mice infected with a less virulent virus was distinct from the Th17 subtype that developed in response to a more virulent virus indicating a role for virus strain in determining the immune response. Slow production of antibody in the nervous system led to delayed virus clearance. Therefore, both virus strain and the host response to infection are important determinants of outcome.

  • Deficiency of the IRE1{alpha}-Autophagy Axis Enhances the Antitumor Effects of the Oncolytic Virus M1 [PublishAheadOfPrint]

  • Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancer cells. M1 is a naturally occurring alphavirus (Togaviridae) which shows potent oncolytic activities to many cancers. Accumulation of unfolded proteins during virus replication leads to a transcriptional/translational response known as the unfolded protein response (UPR), which might counteract the antitumor effect of the oncolytic virus. In this report, we show that either pharmacological or biological inhibition of IRE1aalpha; or PERK, but not ATF6, substantially increases the oncolytic effects of the M1 virus. Moreover, inhibition of IRE1aalpha; blocks M1 virus-induced autophagy, which restricts the antitumor effects of the M1 virus through degradation of viral protein, in glioma cells. In addition, IRE1aalpha; suppression significantly increases the oncolytic effect of M1 virus in an orthotopic glioma model. From a molecular pathology study, we found that IRE1aalpha; is expressed at lower levels in higher grade gliomas, suggesting greater antitumor efficacy of the oncolytic virus M1. Taken together, these findings illustrate a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and the subsequent lysis of tumor cells.

    Importance Although oncolytic virotherapy is showing great promise in clinical applications, not all patients are benefiting. Identifying inhibitory signals in refractory cancer cells for each oncolytic virus would provide a good chance to increase therapeutic effect. Here we describe that infection of the oncolytic virus M1 triggers the unfolded protein response (UPR) and the subsequent autophagy, while blocking the UPR-autophagy axis significantly potentiate the antitumor efficacy of M1 in vitro and in vivo. A survey of cancer tissue banks reveals that IRE1aalpha;, a key element in UPR pathway, is commonly down-regulated in higher grade human gliomas, suggesting favorable application prospects of M1. Our work provides a potential predictor and target for enhancement of the therapeutic effectiveness of the M1 virus. We predict that the mechanism-based combination therapy will promote cancer virotherapy in the future.

  • Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins [PublishAheadOfPrint]

  • Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral non-structural protein, NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon-antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85bbeta;, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone, nor infection with a chimeric bat FLUAV, efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1:p85bbeta; interaction can be re-engineered (in a strain-specific manner) by changing 2-4 NS1 residues (96L, 99M, 100I and 145T) thereby creating a hydrophobic patch. Notably, ameliorated p85bbeta;-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type dependent, but species independent, propagation phenotypes. We hypothesize that bat FLUAV hijack of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit unique energy-use strategies of this flying mammal. Our data expand understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs.

    IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly-described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties, and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

  • A single amino acid substitution within the Paramyxovirus Sendai virus nucleoprotein is a critical determinant for production of IFN-beta-inducing copyback-type defective interfering genomes [PublishAheadOfPrint]

  • One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective-interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon (IFN)-beta during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-non-producing phenotypes, from the stock Sendai virus (SeV) strain Cantell, which is widely used in a number of studies on anti-viral innate immunity as a representative IFN-beta-inducing virus. IFN-beta induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-non-producing clone, probably causing both production of the DI genomes and their formation of a stem--loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.

    IMPORTANCE The type-I interferon (IFN) system is a pivotal defense against infecting RNA viruses, which is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective-interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as a high-level IFN-beta induction and strong inhibition of type-I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes as well as viral--host interactions during innate immunity.

  • Human Cytomegalovirus Replication is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 Through Distinctively Different Mechanisms. [PublishAheadOfPrint]

  • Human Cytomegalovirus (HCMV) is the top viral cause of birth defects worldwide, and current therapies have high toxicity. We previously published that the mTOR-independent autophagy-inducing disaccharide trehalose inhibits HCMV replication in multiple cell types. Here, we examine the mechanism of inhibition and introduce the autophagy inducer SMER28 as an additional inhibitor of HCMV acting through a different mechanism. We find that trehalose induces vacuolation and acidification of vacuoles, and that debris, including debris consistent in appearance with abnormal virions, is present in multivesicular bodies. Trehalose treatment increased levels of Rab7, a protein required for lysosomal biogenesis and fusion, and slightly decreased Rab11, which is associated with recycling endosomes. We also present evidence that trehalose can promote autophagy without altering cellular glucose uptake. We show that SMER28 inhibits HCMV at the level of early protein production and interferes with viral genome replication in a cell-type dependent fashion. Finally, we show that SMER28 treatment does not cause the vacuolation, acidification, or redistribution of Rab7 associated with trehalose treatment and shows only a modest and cell-type dependent effect on autophagy. We propose a model in which the reciprocal effects on Rab7 and Rab11 induced by trehalose contribute to redirection of enveloped virions from the plasma membrane to acidified compartments and subsequent degradation, and SMER28 treatment results in decreased expression of early and late proteins, reducing the number of virions produced without the widespread vacuolation characteristic of trehalose treatment.

    IMPORTANCE There is a need for less toxic HCMV antiviral drugs, and modulation of autophagy to control viral infection is a new strategy that takes advantage of viral dependence on autophagy inhibition. The present study extends our previous work on trehalose by showing a possible mechanism of action and introduces another autophagy-inducing compound, SMER28, as effective against HCMV in several cell types. The mechanism by which trehalose induces autophagy is currently unknown, though our data show that trehalose does not inhibit cellular glucose uptake in cells relevant for HCMV replication, but instead alters virion degradation by promoting acidic vacuolization. The comparison of our cell types and those used by others highlights the cell-type dependent nature of studying autophagy.

  • Human Cytomegalovirus UL111A and US27 gene products enhance the CXCL12/CXCR4 signaling axis via distinct mechanisms [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes lifelong infection in the host. Virus persistence is aided by extensive manipulation of the host immune system, particularly cytokine and chemokine signaling pathways. The HCMV UL111A gene encodes cmvIL-10, an ortholog of human interleukin-10 that has many immunomodulatory effects. We found that cmvIL-10 increased signaling outcomes from human CXCR4, a chemokine receptor with essential roles in hematopoiesis and immune cell trafficking, in response to its natural ligand CXCL12. Calcium flux and chemotaxis to CXCL12 was significantly greater in the presence of cmvIL-10 in monocytes, epithelial cells, and fibroblasts that express CXCR4. CmvIL-10 effects on CXCL12/CXCR4 signaling required the IL-10 receptor and Stat3 activation. Heightened signaling occurred both in HCMV-infected cells as well as in uninfected bystander cells, suggesting that cmvIL-10 may broadly influence chemokine networks by paracrine signaling during infection. Moreover, CXCL12/CXCR4 signaling was amplified in HCMV-infected cells compared to mock-infected cells even in the absence of cmvIL-10. Enhanced CXCL12/CXCR4 outcomes were associated with expression of the virally encoded chemokine receptor US27, and CXCL12/CXCR4 activation was reduced in cells infected with a deletion mutant lacking US27 (TB40/E-mCherry-US27). US27 effects were Stat3-independent but required close proximity to CXCR4 in cell membranes of either HCMV-infected or US27-transfected cells. Thus, HCMV encodes two proteins, cmvIL-10 and US27, that exhibit distinct mechanisms for enhancing CXCR4 signaling. Either individually or in combination, cmvIL-10 and US27 may enable HCMV to exquisitely manipulate CXCR4 signaling to alter host immune responses and modify cell trafficking patterns during infection.

    IMPORTANCE The human chemokine system plays a central role in host defense, as evidenced by the many strategies devised by viruses for manipulating it. Human cytomegalovirus (HCMV) is widespread in the human population, but infection rarely causes disease except in immune compromised hosts. We found that two different HCMV proteins, cmvIL-10 and US27, act through distinct mechanisms to up-regulate the signaling activity of a cellular chemokine receptor, CXCR4. CmvIL-10 is a secreted viral cytokine that affects CXCR4 signaling in both infected and uninfected cells, while US27 is a component of the virus particle and impacts CXCR4 activity only in infected cells. Both cmvIL-10 and US27 promote increased intracellular calcium signaling and cell migration in response to chemokine CXCL12 binding to CXCR4. Our results demonstrate that HCMV exerts fine control over the CXCL12/CXCR4 pathway, which could lead to enhanced virus dissemination, altered immune cell trafficking, and serious health implications for HCMV patients.

  • Structural basis for the inhibition of host gene expression by porcine epidemic diarrhea virus nsp1 [PublishAheadOfPrint]

  • Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the pork industry. The non-structural protein 1 (nsp1) is a characteristic feature of Alpha- and Beltacoronaviruses, which exhibits both functional conservation and mechanistic diversity in inhibiting host gene expression and antiviral responses. However, the detailed structure and molecular mechanisms underlying the Alphacoronavirus nsp1 inhibition of host gene expression remain unclear. Here, we report the first full-length crystal structure of Alphacoronavirus nsp1 from PEDV. The structure displays a six-stranded bbeta;-barrel fold in the middle of two aalpha;-helices. The core structure of PEDV nsp1 shows high similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 and transmissible gastroenteritis virus (TGEV) nsp1, despite its low degree of sequence homology. Using ribopuromycylation and Renilla luciferase reporter assays, we show that PEDV nsp1 can dramatically inhibit general host gene expression. Furthermore, three motifs (67-71 aa, 78-85 aa and 103-110 aa) of PEDV nsp1 create a stable functional region for inhibiting protein synthesis, differing considerably from Betacoronavirus nsp1. These results elucidate the detailed structural basis through which PEDV nsp1 inhibits host gene expression, providing insight into the development of a new attenuated vaccine with nsp1 modifications.


    Porcine epidemic diarrhea virus (PEDV) has led to tremendous economic losses in the global swine industry. PEDV nsp1 plays a crucial role in inhibiting host gene expression, but its functional mechanism remains unclear. Here, we report the first full-length structure of PEDV nsp1 among coronaviruses. The 1.25-AAring; resolution crystal structure of PEDV nsp1 shows high similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) nsp113-128 and transmissible gastroenteritis virus (TGEV) nsp11-104, despite a lack of sequence homology. Structural and biochemical characterization demonstrated that PEDV nsp1 possesses a stable functional region for inhibition of host protein synthesis, which is formed by loops at residues 67-71, 78-85 and 103-110. The different functional regions between PEDV nsp1 and SARS-CoV nsp1 may explain their distinct mechanisms. Importantly, our structural data is conducive to understanding the mechanism of PEDV nsp1 inhibiting the expression of host genes and may aid in the development of a new attenuated vaccine.

  • Neutralizing activity of broadly neutralizing anti-HIV-1 antibodies against clade B clinical isolates produced in peripheral blood mononuclear cells [PublishAheadOfPrint]

  • Recently discovered broadly neutralizing anti-HIV-1 antibodies (bNAbs) demonstrate extensive breadth and potency against diverse HIV-1 strains, and represent a promising approach for the treatment and prevention of HIV-1 infection. The breadth and potency of these antibodies have primarily been evaluated using panels of HIV-1 Env-pseudotyped viruses produced in 293T cells expressing molecularly cloned Envs. Here we report on the ability of 5 bNAbs currently in clinical development to neutralize circulating primary HIV-1 isolates derived from peripheral blood mononuclear cells (PBMC), and compare the results to the pseudovirus panels used to characterize the bNAbs. The 5 bNAbs demonstrated significantly reduced breadth and potency when tested against clinical isolates produced in PBMC compared to their performance against Env-pseudotyped viruses. The magnitude of this difference in neutralizing activity varied depending on the antibody epitope. Glycan-targeting antibodies showed differences of only 3 to 4-fold while the MPER-targeting antibody 10E8 showed a nearly 100-fold decrease in activity between published Env-pseudotyped virus panels and PBMC-derived primary isolates. Utilizing clonal PBMC-derived primary isolates and molecular clones, we determined that the observed discrepancy in bNAb performance is due to the increased sensitivity to neutralization exhibited by 293T-produced Env-pseudotyped viruses. We also found that while full-length molecularly cloned viruses produced in 293T cells exhibit increased sensitivity to neutralization compared to PBMC-derived viruses, Env-pseudotyped viruses produced in 293T cells generally exhibit even greater sensitivity to neutralization. As the clinical development of bNAbs progresses, it will be critical to determine the relevance of each of these in vitro neutralization assays to in vivo antibody performance.


    Novel therapeutic and preventative strategies will be needed to contain the HIV-1 epidemic. Antibodies with exceptional neutralizing activity against HIV-1 may provide several advantages to traditional HIV drugs, including an improved side effect profile, reduced dosing frequency, and immune enhancement. The activity of these antibodies has been established in vitro utilizing HIV-1 Env-pseudotyped viruses derived from circulating viruses but produced in 293T cells by pairing Envs with a backbone vector. We tested PBMC-produced circulating viruses against 5 anti-HIV-1 antibodies currently in clinical development. We found that the activity of these antibodies is significantly reduced against PBMC isolates as compared to 293T Env-pseudotyped viruses. This decline varied among the antibodies tested, with some demonstrating moderate reductions in activity while others showing almost 100-fold reduction. As the development of these antibodies progresses, it will be critical to determine how the results of different in vitro tests correspond to performance in the clinic.

  • Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication [PublishAheadOfPrint]

  • Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase and polymerase aalpha; are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this study provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.

    IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red-cell aplasia. In fetuses, B19V infection can result in non-immune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression, and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome, and played a critical role in viral DNA replication. By contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

  • Mammalian adaptation of an avian influenza A virus involves stepwise changes in NS1 [PublishAheadOfPrint]

  • Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral non-structural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host-species barrier.

    We characterised the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionary distinct NS1s counteract the IFN response using different and mutually exclusive mechanisms: while the NS1s of early EIVs block general gene expression by binding to the cellular polyadenylation specific factor 30 (CPSF30), NS1s from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and became rapidly selected during EIV evolution, highlighting the importance of evolutionary processes on immune evasion mechanisms during IAV adaptation. [150 words]

    IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species, and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remains unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that has successfully established a new lineage in horses in the early 1960, and is currently circulating worldwide in the equine population. Here we analysed the molecular evolution of the virulence factor non-structural protein 1 (NS1) and show that NS1s from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

  • Molecular dynamics and mode of transmission of Koala Retrovirus (KoRV) as it invades and spreads through a wild Queensland koala population [PublishAheadOfPrint]

  • The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV; KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes requires population-level studies of their prevalence and diversity, especially when co-infected in the same population, and investigations of their modes of transmission in the wild. Towards this end, we studied a wild Queensland koala population of 290 animals over a five year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope gene protein sequence. By comparison, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data.

    IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form, and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B infected/co-infected animals and its linkage to chlamydial disease, neoplasia, viral evolution and spread.

  • Maintenance of AP-2 Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses [PublishAheadOfPrint]

  • Nef-specific CD8+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+ restricted Nef195-203MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28, and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or MHC-I, suggesting selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2 mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically.


    A rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the SIV Nef protein are capable of better control of the virus and that, as the virus evolves to escape this response, it does so at a cost to specific functions performed by the Nef protein. Our results help to understand how the virus can be controlled by an immune response, which could help in designing effective vaccines.

  • HSV-1 DNA Polymerase RNase H Activity Acts in a 3' -5' Direction and is Dependent on the 3' -5' Exonuclease Active Site [PublishAheadOfPrint]

  • The catalytic subunit (Pol) of herpes simplex virus-1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA:DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3' -5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3' -5' exonuclease-deficient mutant, D368A, for DNA polymerase activity, 3' -5' exonuclease activity, and RNase H activity in vitro. Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity, but was drastically impaired for 3' -5' exonuclease activity, with no activity detected even at high enzyme to DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3' or 5' terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA:DNA hairpin template, but only detectably with a 3' RNA terminus in a 3' -5' direction, and at a rate slower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3' -5' exonuclease activity, and that this activity prefers DNA degradation over degradation of RNA from RNA:DNA hybrids.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) is a member of the Herpesviridae family of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA Polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5' -3' RNase H activity intrinsic to this enzyme that could remove RNA primers from Okazaki fragments has been particularly controversial. In this study, we were unable to identify RNase H activity of HSV-1 DNA polymerase on RNA:DNA hybrids with 5' RNA termini. We detected RNase H activity on hybrids with 3' termini, but this was due to 3' -5' exonuclease. Thus, HSV-1 is unlikely to use this method to remove RNA primers during DNA replication, but may use pathways similar to those used in eukaryotic Okazaki fragment maturation.

  • A trimeric HIV-1 envelope gp120 immunogen induces potent and broad anti-V1V2 loop antibodies against HIV-1 in rabbits and rhesus macaques [PublishAheadOfPrint]

  • Trimeric HIV-1 envelope (Env) immunogens are attractive due to their ability to display quaternary epitopes targeted by broadly neutralizing antibodies while obscuring unfavorable epitopes. Results from the RV144 trial highlighted the importance of vaccine induced HIV-1 Env V1V2 directed antibodies, with key regions of the V2 loop as targets for vaccine-mediated protection. We recently reported that a trimeric JRFL-gp120 immunogen, generated by inserting a N-terminal trimerization domain in the V1 loop region of a cyclically permuted gp120 (cycP-gp120), induces neutralizing activity against multiple tier-2 HIV-1 isolates in guinea pigs in a DNA prime/protein boost approach. Here, we tested the immunogenicity of cycP-gp120 in a protein prime/boost approach in rabbits and as a booster immunization to DNA/MVA vaccinated rabbits and rhesus macaques. In rabbits, two cycP-gp120 protein immunizations induced 100-fold higher titers of high avidity gp120-specific IgG compared to two gp120 immunizations, with four total gp120 immunizations being required to induce comparable titers. CycP-gp120 also induced markedly enhanced neutralizing activity against tier-1A and -1B HIV-1 isolates, substantially higher binding and breadth to gp70-V1V2 scaffolds derived from a multi-clade panel of global HIV-1 isolates, and antibodies targeting key regions of the V2-loop region associated with reduced risk of infection in RV144. Similarly, boosting MVA or DNA/MVA primed rabbits or rhesus macaques with cycP-gp120 showed a robust expansion of gp70-V1V2-specific IgG, neutralization breadth to tier-1B HIV-1 isolates and ADCC activity. These results demonstrate that cycP-gp120 serves as a robust HIV Env immunogen that induces broad anti-V1V2 antibodies and promotes neutralization breadth against HIV-1.

    IMPORTANCE Recent focus in HIV-1 vaccine development has been the design of trimeric HIV-1 envelope (Env) immunogens that closely resemble native HIV-1 Env, with a major goal being the induction of broadly cross-reactive neutralizing antibodies (bNAbs). While the generation of bNAbs are considered a gold-standard in vaccine-induced antibody responses, results from the RV144 trial showed that non-neutralizing antibodies directed towards the V1V2-loop of HIV-1 gp120, specifically the V2 loop region, were associated with decreased risk of infection demonstrating the need for the development of Env immunogens that induce a broad anti-V1V2 antibody response. In this study, we show that a novel trimeric gp120 protein, cycP-gp120, generates high titers of high avidity and broadly cross-reactive anti-V1V2 antibodies, a result not found in animals immunized with monomeric gp120. These results reveal the potential of cycP-gp120 as a vaccine candidate to induce antibodies associated with reduced risk of HIV-1 infection in humans.

  • Identification of novel structural determinants in MW965 Env that regulate the neutralization phenotype and conformational masking potential of primary HIV-1 isolates [PublishAheadOfPrint]

  • The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier-1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possessed strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env were responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, ConC, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while having no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identified novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates.


    A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 viruses would provide important information about the natural structural and conformational diversity of HIV-1 Env, and how this affects neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains, and demonstrated that the sequences at these positions differentially affected the presentation of epitopes recognized by different classes of standard and conformational-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate neutralization phenotype, and provide tools for controlling Env conformation that could have applications both for structural studies and in vaccine design.

  • HLA class I-mediated HIV-1 control in Vietnamese infected with HIV-1 subtype A/E [PublishAheadOfPrint]

  • HIV-1-specific cytotoxic T cells (CTLs) play an important role in the control of HIV-1 subtype B or C infection. However, the role of CTLs in HIV-1 subtype A/E infection still remains unclear. Here we investigated the association of HLA class I alleles with clinical outcome in treatment-naïve Vietnamese infected with subtype A/E. We found that HLA-C*12:02 was significantly associated with lower pVL and higher CD4 count and that the HLA-A*29:01-B*07:05-C*15:05 haplotype was significantly associated with higher pVL and lower CD4 count as compared to individuals without these respective genotypes. Nine Pol and three Nef mutations were associated with at least one HLA allele in the HLA-A*29:01-B*07:05-C*15:05 haplotype, where a strong negative correlation between the number of HLA-associated Pol mutations and CD4 count as well as a positive correlation with pVL in individuals with these HLA alleles were observed. The results suggest that the accumulation of mutations selected by CTLs restricted by these HLA alleles affect HIV control.


    Most previous studies on HLA association with disease progression after HIV-1 infection have been performed in cohorts infected with HIV-1 subtypes B and C, whereas few such population-based studies have reported in cohorts infected with the Asian subtype A/E virus. In this study, we analyzed the association of HLA class I alleles with clinical outcomes in 536 HIV-1 subtype A/E infected Vietnamese individuals. We found that HLA-C*12:02 is protective while the HLA haplotype HLA-A*29:01-B*07:05-C*15:05 is deleterious. The individuals with HIV-1 mutations associated with at least one of the HLA alleles in the deleterious HLA haplotype had higher plasma viral load and lower CD4 counts than those without the mutations, suggesting viral adaptation and escape from HLA-mediated immune control. The present study identified a protective allele and a deleterious haplotype in the subtype A/E infection, which are different from those identified in cohorts infected with HIV-1 subtypes B and C.

  • The partitivirus HetPV13-an1 mediates severe growth debilitation and major alterations in the gene expression of a fungal forest pathogen [PublishAheadOfPrint]

  • The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the Boreal forest region. In this study we showed that the alphapartitivirus Heterobasidion partitivirus 13 from H. annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, from which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and H. parviporum. The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing media, while only two of six H. annosum isolates tested showed significant debilitation. Based on RT-qPCR analysis, the response towards HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum. We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.


    A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of 800 million in Europe. We used virus curing and re-introduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a slow growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

  • Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication [PublishAheadOfPrint]

  • Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.

    IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.

  • Axl can serve as entry factor for Lassa virus depending on the functional glycosylation of dystroglycan [PublishAheadOfPrint]

  • Fatal infection with the highly pathogenic Lassa virus (LASV) is characterized by extensive viral dissemination, indicating broad tissue tropism. The major cellular receptor for LASV is the highly conserved extracellular matrix receptor dystroglycan (DG). Binding of LASV depends on DG's tissue-specific post-translational modification with the unusual O-linked polysaccharide matriglycan. Interestingly, functional glycosylation of DG does not always correlate with viral tropism observed in vivo. The broadly expressed phosphatidylserine (PS) receptors Axl and Tyro3 were recently identified as alternative LASV receptor candidates. However, their role in LASV entry is not entirely understood. Here we examined LASV receptor candidates in primary human cells and found co-expression of Axl with differentially glycosylated DG. To study LASV receptor use in the context of productive arenavirus infection, we employed recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rLCMV-LASVGP) as validated BSL2 model. We confirm and extend previous work, showing that Axl can contribute to LASV entry in absence of functional DG using "apoptotic mimicry", similar to other enveloped virus. We further show that Axl-dependent LASV entry requires receptor activation and involves a pathway resembling macropinocytosis. Axl-mediated LASV entry is facilitated by heparan sulfate and critically depends on the late endosomal protein LAMP-1 as intracellular entry factor. In endothelial cells expressing low levels of functional DG, both receptors are engaged by the virus and can contribute to productive entry. In sum, we characterize the role of Axl in LASV entry and provide a rationale to target Axl in anti-viral therapy.


    The highly pathogenic arenavirus Lassa (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor dystroglycan (DG) is ubiquitously expressed, virus binding critically depends on DG's post-translational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as alternative LASV receptor candidate, but its role in LASV entry is unclear. Here we investigated the exact role of Axl in LASV entry as a function of DG's post-translational modification. We found that in absence of functional DG, Axl can mediate LASV entry via "apoptotic mimicry". Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale to target Axl in anti-viral therapy.

  • Human Norovirus NS3 has RNA Helicase and Chaperoning Activities [PublishAheadOfPrint]

  • RNA remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions, and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-stranded RNA viruses in the family Caliciviridae, and constitute a significant and potentially fatal threat to human health. Here we report that protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA chaperoning activity that can remodel structured RNAs and facilitate strand-annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound and, more importantly, that it reduces the replication of norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA remodeling activities encoded by Caliciviridae, and highlight the functional significance of NS3 in noroviral life cycle.

    IMPORTANCE Noroviruses are a diverse group of positive-stranded RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to associate with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein NS3 not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. And NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for anti-noroviral drug development.

  • The three-fold axis of the HIV-1 capsid lattice is the species-specific binding interface for TRIM5{alpha} [PublishAheadOfPrint]

  • Rhesus TRIM5aalpha; (rhTRIM5aalpha;) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5aalpha; to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remains largely unknown. The human orthologue of TRIM5aalpha; (hsTRIM5aalpha;) fails to block infection by HIV-1 both in vivo and in vitro. This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5aalpha; B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the three-fold axis of symmetry. The results demonstrate that TRIM5aalpha; binding involves multiple capsid molecules along the two-fold and three-fold interfaces between hexamers and indicate that the binding interface at the three-fold axis contributes to the well-established differences in restriction potency between TRIM5aalpha; orthologues.

    SIGNIFICANCE STATEMENT TRIM5aalpha; is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5aalpha; restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5aalpha; confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5aalpha; to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5aalpha;.

  • Structure of the paramyxovirus PIV5 nucleoprotein in complex with an amino-terminal peptide of the phosphoprotein [PublishAheadOfPrint]

  • Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped viruses with a non-segmented negative strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent non-specific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N-terminus of P (P50) at 2.65 AAring;. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown as an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends towards the RNA binding site to inhibit N oligomerization and hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.


    Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of CTD and the importance of domain movement for the conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.

  • The neutralizing linear epitope of human herpesvirus-6A glycoprotein B does not affect virus infectivity. [PublishAheadOfPrint]

  • Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the virus's growth. Previously, we reported that a neutralizing antibody (Mab) called 87-y-13 specifically reacts with HHV-6A gB and identified its epitope residue at 347 asparagine (Asn) on gB. In this study, we examined whether the epitope recognized by the neutralizing Mab is essential for HHV-6A infection. We constructed HHV-6A BAC genomes harboring substitutions at 347Asn, namely HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild-type and their revertants, and Mab 87-y-13 could not inhibit infection of either mutant. In a cell--cell fusion assay, Asn at 347 on gB was found to be non-essential for cell--cell fusion. In addition, in building an HHV-6A gB homology model, we found that its epitope is located on domain II of gB and accessible to the solvent. These results indicate that Asn at 347, the linear epitope of the neutralizing Mab, does not affect HHV-6A infectivity.

    IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize the virus entry. Human herpesvirus 6A (HHV-6A) also encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (Mab) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, in regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to the solvent, suggesting that it might be a target of the immune system.

  • Influenza A virus reassortment is limited by anatomical compartmentalization following co-infection via distinct routes [PublishAheadOfPrint]

  • Exchange of gene segments through reassortment is a major feature of influenza A virus evolution and frequently contributes to the emergence of novel epidemic, pandemic and zoonotic strains. It has long been evident that viral diversification through reassortment is constrained by genetic incompatibility between divergent parental viruses. In contrast, the role of virus-extrinsic factors in determining the likelihood of reassortment has remained unclear. To evaluate the impact of such factors in the absence of confounding effects of segment mismatch, we previously reported an approach in which reassortment between wild-type (wt) and genetically tagged variant (var) viruses of the same strain is measured. Here, using wt/var systems in the A/Netherlands/602/2009 (pH1N1) and A/Panama/2007/99 (H3N2) strain backgrounds, we have tested whether inoculation of parental viruses into distinct sites within the respiratory tract limits their reassortment. Using a ferret model, matched parental viruses were either co-inoculated intranasally, or one virus was instilled intranasally while the second was instilled intratracheally. Dual intranasal inoculation resulted in robust reassortment for wt/var viruses of both strain backgrounds. In contrast, when infections were initiated simultaneously at distinct sites, strong compartmentalization of viral replication was observed and minimal reassortment was detected. The observed lack of viral spread between upper and lower respiratory tissues may be attributable to localized exclusion of super-infection within the host, mediated by innate immune responses. Our findings indicate that dual infections in nature are more likely to result in reassortment if viruses are seeded into similar anatomical locations and have matched tissue tropisms.


    Genetic exchange between influenza A viruses (IAVs) through reassortment can facilitate the emergence of antigenically drifted seasonal strains and plays a prominent role in the development of pandemics. Typical human influenza is concentrated in the upper respiratory tract; however, lower respiratory tract (LRT) infection is an important feature of severe cases, which are more common in the very young, the elderly, and individuals with underlying conditions. In addition to host factors, viral characteristics and mode of transmission can also increase the likelihood of LRT infection: certain zoonotic IAVs are thought to favor the LRT and transmission via small droplets allows direct seeding into lower respiratory tissues. Toward gauging the likelihood of reassortment in co-infected hosts, we assessed the extent to which initiation of infection at distinct respiratory sites impacts reassortment frequency. Our results reveal that spatially distinct inoculation results in anatomical compartmentalization of infection, which in turn strongly limits reassortment.

  • Suppression of Zika virus infection and replication in endothelial cells and astrocytes by PKA inhibitor PKI 14-22 [PublishAheadOfPrint]

  • The recent outbreak of Zika virus (ZIKV), a re-emerging flavivirus, and its associated neurological disorders, such as Guillain-Barreeacute; (GB) syndrome and microcephaly, have generated an urgent need for developing effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors for ZIKV replication. Because several pathways, including AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors or agonists of these pathways for their effects on ZIKV replication. We identified PKA inhibitor, PKI 14-22 (PKI), as a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both African and Asian/American lineages with high efficiency and minimal cytotoxicity. While ZIKV infection did not induce PKA activation, endogenous PKA activity was essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited other unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the post-entry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication, which could be further explored for future therapeutic application.


    There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a re-emerging flavivirus associated with neurological disorders including Guillain-Barreeacute; (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified PKA inhibitor PKI 14-22 (PKI) as a potent inhibitor of ZIKV replication. We have shown that PKI effectively suppresses the replication of ZIKV of all the strains tested with minimal cytotoxicity in human endothelial cells and astrocytes, two key cell types of ZIKV infection. Furthermore, we have shown that PKI inhibits ZIKV negative-sense RNA synthesis and viral protein translation. This study has identified a potent inhibitor of ZIKV infection, which could be further explored for future therapeutic application.

  • Tetraspanin CD63 bridges autophagic and endosomal processes to regulate exosomal secretion and intracellular signaling of Epstein-Barr virus LMP1. [PublishAheadOfPrint]

  • The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations, and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes, and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of crosstalk between endosomes and autophagosomes, and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion, and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity, in both non-infected and virally-infected cells.


    The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein LMP1 into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate the key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insight into the complex intersection of cellular secretory and degradative mechanisms, and the implications of these processes in viral replication.

  • Minor capsid protein L2 polytope induces broad protection against oncogenic and mucosal human papillomaviruses [PublishAheadOfPrint]

  • The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. Wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1 based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV types 16, 31, and 51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here we report the development of a thermostable thioredoxin based single peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types we have analyzed plus the low risk HPV types 6 and 11 and a number of cutaneous HPVs.


    Infections by a large number of human papillomaviruses lead to malignant and non-malignant disease. Current commercial vaccines based on virus-like particles effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so called neutralization epitopes within its N-terminus. We designed polytopes comprising the L2 epitope amino acids 20-38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile achaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising an heptamerized L2 8mer thioredoxin single peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

  • Experimental adaptive evolution of SIVcpz to pandemic HIV-1 using a humanized mouse model [PublishAheadOfPrint]

  • HIV-1, the causative agent of AIDS, is originated from SIVcpz, the chimpanzee precursor of the human virus, approximately 100 years ago. This indicates that HIV-1 has emerged through the cross-species transmission of SIVcpz from chimpanzees to humans. However, it remains unclear how SIVcpz has evolved into pandemic HIV-1 in humans. To address this question, we inoculated three SIVcpz (MB897, EK505, and MT145), four pandemic HIV-1 (NL4-3, NLCSFV3, JRCSF and AD8) and 2 non-pandemic HIV-1 (YBF30 and DJO0131) strains. Humanized mice infected with SIVcpz strain MB897, a virus phylogenetically similar to pandemic HIV-1, exhibited a comparable peak viral load to that of mice infected with pandemic HIV-1, while peak viral loads of mice infected with SIVcpz strains EK505 or MT145 as well as non-pandemic HIV-1 strains were significantly lower. These results suggest that SIVcpz strain MB897 is pre-adapted to humans when compared to the other SIVcpz strains. Moreover, viral RNA sequencing of MB897-infected humanized mice identified a nonsynonymous mutation in env, G413R substitution in gp120. The infectivity of the gp120 G413R mutant of MB897 was significantly higher than that of parental MB897. Furthermore, we demonstrated that the gp120 G413R mutant of MB897 augments the capacity for viral replication in both in vitro cell cultures and humanized mice. Taken together, this is the first experimental investigation to use an animal model to demonstrate a gain-of-function evolution of SIVcpz into pandemic HIV-1.


    From the mid-20th century, humans are exposed to the menace of viral infectious diseases such as SARS coronavirus, Ebola virus and Zika virus. These outbreaks of emerging/re-emerging viruses can be triggered by cross-species viral transmission from wild animals to humans or zoonoses. HIV-1, the causative agent of AIDS, was emerged by the cross-species transmission of SIVcpz, the HIV-1 precursor in chimpanzee, around 100 years ago. However, the process by which SIVcpz evolved to become HIV-1 in humans remains unclear. By using a hematopoietic stem cell-transplanted humanized mouse model, here we experimentally recapitulate the evolutionary process of SIVcpz to become HIV-1. We provide evidence suggesting that a strain of SIVcpz, MB897, has pre-adapted to infect humans comparing to other SIVcpz strains. We further demonstrate a gain-of-function evolution of SIVcpz in infected humanized mice. Our study reveals that pandemic HIV-1 has emerged through at least two steps: preadaptation and subsequent gain-of-function mutations.

  • HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment is Required for Particle Incorporation [PublishAheadOfPrint]

  • The HIV-1 envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-Family Interacting Protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxx endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC, and supports a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C and CT-dependent outward sorting to the particle assembly site on the plasma membrane.


    The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reach the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles, and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

  • Control of HTLV-1 Infection by Eliminating Envelope Protein-Positive Cells with Recombinant Vesicular Stomatitis Viruses Encoding HTLV-1 Primary Receptor [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia (ATL), which is frequently resistant to current available therapies and has a very poor prognosis. To prevent the development of ATL among carriers it is important to control HTLV-1nndash;infected cells in infected individuals. Therefore, the establishment of novel therapies with drugs specifically targeting infected cells is urgently required. This study aimed to develop a potential therapy by generating recombinant vesicular stomatitis viruses (rVSVs) that lack an envelope glycoprotein G and instead encode HTLV-1 receptor(s) with human glucose transporter 1 (GLUT1), neuropilin 1 (NRP1), or heparan sulfate proteoglycans (HSPGs) including syndecan 1 (SDC1), designated as VSVG-GL, VSVG-NP, or VSVG-SD, respectively. In an attempt to enhance the infectivity of rVSV against HTLV-1nndash;infected cells, we also constructed rVSVs with a combination of two or three receptor genes, designated as VSVG-GLN and VSVG-GLNS, respectively. The current study demonstrated VSVG-GL, VSVG-NP, VSVG-GLN, and VSVG-GLNS have tropism for HTLV-1 envelope (Env) expressing cells. Notably, the inoculation of VSVG-GL or VSVG-NP significantly eliminated HTLV-1nndash;infected cells in the culture conditions. Furthermore, in an HTLV-1nndash;infected humanized mouse model, VSVG-NP was capable of efficiently preventing HTLV-1nndash;induced leukocytosis in the periphery and eliminating HTLV-1nndash;infected Env-expressing cells in the lymphoid tissues. In summary, an rVSV engineered to express HTLV-1 primary receptor, especially human NRP1, may represent a drug candidate that has potential for the development of unique virotherapy against HTLV-1 de novo infection.


    Although several anti-ATL therapies are currently available, ATL is still frequently resistant to therapeutic approaches and its prognosis remains poor. Control of de novo HTLV-1 infection or expansion of HTLV-1nndash;infected cells in the carrier holds considerable promise for the prevention of ATL development. In this study, we developed rVSVs that specifically target and kill HTLV-1 Env-expressing cells (not ATL cells, which generally do not express Env in vivo) through replacement of the G gene with HTLV-1 receptor gene(s) in the VSV genome. Notably, an rVSV engineered to express human NRP1 controlled the number of HTLV-1nndash;infected Env-expressing cells in vitro and in vivo, suggesting the present approach may be a promising candidate for novel anti-HTLV-1 virotherapy in HTLV-1 carriers, including as a prophylactic treatment against the development of ATL.

  • Structural insight into a human neutralizing antibody against influenza virus H7N9 [PublishAheadOfPrint]

  • Since its first emergence in East China in early 2013, many cases of avian influenza A H7N9 have been reported. The disease has extended to 22 provinces in mainland China and some surrounding areas. Strategies to combat viral infection are urgently needed. We previously isolated a human monoclonal antibody, HNIgGA6, that neutralized H7N9 virus both in vitro and in vivo. In this study, we determined the crystal structure of viral hemagglutinin (HA) globular head bound to the Fab fragment of HNIgGA6. The crystal structure shows that the tip of the HNIgGA6 heavy-chain complementarity determining region 3 (HCDR3) directly interposes into the receptor binding site (RBS) and mimics, in many respects, the interaction of the sialic acid receptor. Three residues atY98, H183 and E190, that are critical to human cellular receptor binding, are also essential for HNIgGA6 recognition. At meanwhile, dual mutations at V186G and L226Q in RBS were able to disrupt viral HA1 binding with the antibody. Our study provides a better understanding of the mechanism for protective antibody recognition and sound foundation for the design of therapeutic drugs and vaccines against H7N9 influenza.


    Neutralization by antibody is one of the most important mechanisms for a host to defend against viral infections. Human-originated antibody HNIgGA6 was generated in response to the natural infectious H7N9 virus and had potential use for suppression of H7N9 infection, with possible therapeutic implications. The crystal structure of HNIgGA6/HA1 complex provided new insight into the protective immune response to H7N9 virus in humans, as well as opinion for the development of effective H7N9 pandemic vaccines and antiviral molecules.

  • Subcellular localization and functional characterization of GII.4 norovirus-encoded NTPase [PublishAheadOfPrint]

  • The genotype GII.4 variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded non-structural proteins (including Nterm, NTPase, P22, VPg, Pro and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (or designated as GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or non-vesicular textures in the cell cytoplasm, and the non-vesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-aa region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. Particularly, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase, which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the genogroup I (GI) HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as Nterm and P22, but not VPg, Pro and RdRp in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail or head-to tail configuration. More importantly, we demonstrate that GII-NTPase possesses a pro-apoptotic activity, which can be further enhanced by coexpression with Nterm or P22.


    Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral non-structural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a genogroup I (GI) strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate i) vesicle formation, ii) ER colocalization, iii) the interaction with two another non-structural proteins including Nterm and P22, iv) the formation of homo-dimers or homo-oligomers, and v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

  • Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection [PublishAheadOfPrint]

  • HIV-1 elite controllers (EC) maintain undetectable viral load (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes including a proportion loosing HIV-1 control over time. In this work we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, versus persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1-infection with a longitudinal retrospective study design. Gag-specific T-cell response was assessed by in vitro intracellular poly-cytokine production quantified by flow cytometry. Viral diversity and sequence-dating were performed in proviral DNA by PCR amplification at limiting dilution in env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity and high proinflammatory cytokines levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r=-0.8; p=0.02). RANTES was a potential biomarker of transient control. This study identified, virological and immunological factors including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in a better clinical management of these individuals and in the development of future curative approaches.

    IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work we found differences in virological and immune factors including soluble inflammatory biomarkers between subjects with persistent control of viral replication and EC that will loss the virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose the natural control of viral replication, and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV-infection.

  • A novel Sulfolobus virus with an exceptional capsid architecture [PublishAheadOfPrint]

  • A novel archaeal virus, denoted Sulfolobus ellipsoid virus 1 (SEV1), was isolated from an acidic hot spring in Costa Rica. The morphologically unique virion of SEV1 contains a protein capsid with 16 regularly spaced striations and an 11-nm-thick envelope. The capsid exhibits an unusual architecture in which the viral DNA, probably in the form of a nucleoprotein filament, wraps around the longitudinal axis of the virion in a plane to form a multilayered disk-like structure with a central hole, and 16 of these structures are stacked to generate a spool-like capsid. SEV1 harbors a linear double-stranded DNA genome of ~23 kb, which encodes 38 predicted open reading frames (ORFs). Among the few ORFs with a putative function is a gene encoding a protein-primed DNA polymerase. Six-fold symmetrical virus-associated pyramids (VAPs) appear on the surface of the SEV1-infected cells, which are ruptured to allow the formation of a hexagonal opening and subsequent release of the progeny virus particles. Notably, the SEV1 virions acquire the lipid membrane in the cytoplasm of the host cell. The lipid composition of the viral envelope correlates with that of the cell membrane. These results suggest the use of a unique mechanism by SEV1 in membrane biogenesis.

    IMPORTANCE Investigation of archaeal viruses has greatly expanded our knowledge of the virosphere and its role in the evolution of life. Here we show that Sulfolobus ellipsoid virus 1 (SEV1), an archaeal virus isolated from a hot spring in Costa Rica, exhibits a novel viral shape and an unusual capsid architecture. The SEV1 DNA wraps multiple times in a plane around the longitudinal axis of the virion to form a disk-like structure, and 16 of these structures are stacked to generate a spool-like capsid. The virus acquires its envelope intracellularly and exits the host cell by creating a hexagonal hole on the host cell surface. These results shed significant light on the diversity of viral morphogenesis.

  • Syrian hamster as an animal model for the study of human influenza virus infection [PublishAheadOfPrint]

  • Ferrets and mice are frequently used as animal models for influenza research. However, ferrets are demanding in terms of housing space and handling, whereas mice are not naturally susceptible to infection with human influenza A or B viruses. Therefore, prior adaptation of human viruses is required for their use in mice. In addition, there are no mouse-adapted variants of the recent H3N2 viruses, because these viruses do not replicate well in mice. In this study, we investigated the susceptibility of Syrian hamsters to influenza viruses with a view to using them as an alternative animal model to mice. We found that hamsters are sensitive to influenza viruses, including the recent H3N2 viruses, without adaptation. Although the hamsters did not show weight loss or clinical signs of H3N2 virus infection, we observed pathogenic effects in the respiratory tracts of the infected animals. All of the H3N2 viruses tested replicated in the respiratory organs of the hamsters, and some of them were detected in the nasal washes of infected animals. Moreover, a pdm09 and a seasonal H1N1 virus, as well as one of the two H3N2 viruses, but not a type B virus, were airborne transmissible in these hamsters. Hamsters thus have potential as a small animal model for the study of influenza virus infection, including studies of the pathogenicity of H3N2 viruses and other strains, as well as H1N1 virus transmission studies.

    IMPORTANCE We found that Syrian hamsters are susceptible to human influenza viruses, including the recent H3N2 viruses, without adaptation. We also found that a pdm09 and a seasonal H1N1 virus, as well as one of the H3N2 viruses, but not a type B virus tested are airborne transmitted in these hamsters. Syrian hamsters thus have potential as a small animal model for the study of human influenza viruses.

  • Mutations in the PA Protein of Avian H5N1 Influenza Viruses Affect Polymerase Activity and Mouse Virulence [PublishAheadOfPrint]

  • To study the influenza viral determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012; QT1480) and 2013 (A/duck/Vietnam/QT1728/2013; QT1728) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans.

    IMPORTANCE H5N1 influenza viruses cause severe infections in humans with a case fatality rate that exceeds 50%. The factors that determine the high virulence of these viruses in humans are not fully understood. Here, we identified two amino acid changes in the viral polymerase PA protein that affect the activity of the viral polymerase complex and virulence in mice. Infection with viruses possessing these amino acid changes may pose an increased risk to humans.

  • Autographa californica Nucleopolyhedrovirus ac75 is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation [PublishAheadOfPrint]

  • Autographa californica nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene of unknown function. In this study, we constructed an ac75 knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms of 18 and 15 kDa of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was predominantly localized in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase-separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm, at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identified ac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles.

    IMPORTANCE During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identified ac75 as a second gene, in addition to ac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

  • Nucleic acid polymers are active against Hepatitis Delta Virus infection in vitro. [PublishAheadOfPrint]

  • In this study, an in vitro infection model for the hepatitis delta virus (HDV) was used to evaluate the antiviral effects of phosphorothioate nucleic acid polymers (NAPs) and investigate their mechanism of action. The results show that NAPs inhibit HDV infection at less than 4 micromolar concentrations in cultures of differentiated human hepatoma cells. NAPs were shown to be active at viral entry, but inactive post entry on HDV RNA replication. Inhibition was independent of the NAPs nucleotide sequence, but dependent on both size and amphipathicity of the polymer. NAPs antiviral activity was effective against HDV virions bearing the main hepatitis B virus (HBV) immune escape substitutions (D144A and G145R) and was pangenomic with regard to HBV envelope proteins. Furthermore, similar to immobilized heparin, immobilized NAPs could bind HDV particles suggesting that entry inhibition was due, at least in part, to preventing attachment of the virus to cell surface glycosaminoglycans. The results document NAPs as a novel class of antiviral compounds that can prevent HDV propagation.


    HDV infection causes the most severe form of viral hepatitis in humans and one of the most difficult to cure. Currently, treatments are limited to long-term administration of interferon at high doses, which provide only partial efficacy. There is thus an urgent need for innovative approaches to identify new antiviral against HDV. The significance of our study is in demonstrating that nucleic acid polymers (NAPs) are active against HDV by targeting the envelope of HDV virions. In an in vitro infection assay, NAPs activity was recorded at less than 4 micromolar concentrations in the absence of cell toxicity. Furthermore, the fact that NAPs could block HDV at viral entry, suggest their potential to control the spread of HDV in a chronically HBV-infected liver. In addition, NAPs anti-HDV activity was pangenomic with regard to HBV envelope proteins and not circumvented by HBsAg substitutions associated with HBV immune escape.

  • The cellular chaperone heat shock protein 90 is required for foot-and-mouth disease virus capsid precursor processing and assembly of capsid pentamers [PublishAheadOfPrint]

  • Productive picornavirus infection requires the hijack of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerisation of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and post-translational modification of the capsid precursor (P1-2A) by viral and cellular enzymes, and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90), impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titre by more than ten-fold while not affecting the activity of a sub-genomic replicon indicating that translation and replication of viral RNA were unaffected by the drug.

    IMPORTANCE Foot-and-mouth disease virus (FMDV), of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates, or the immediate co-translational modification of the capsid precursor protein. Here we describe a system to analyse the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur which could be the bases for a novel antiviral control mechanism for FMDV.

  • The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. RSV F comprises a trimer of disulfide bonded F1 and F2 subunits that is present on the virion surface in a llsquo;metastable' pre-fusion state. This pre-fusion form is readily triggered to undergo refolding to bring two heptad repeats (HRA and HRB) into close proximity to form a six-helix bundle that stabilizes the post-fusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75-97), an amphipathic aalpha;-helix that lies at the interface of the pre-fusion F trimer and is a major structural feature of the F2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Herein we propose a model in which HRC plays a stabilizing role within the globular head for the pre-fusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the post-fusion state.


    RSV is recognized as the most important viral pathogen amongst pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of pre-fusion stabilized F protein based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here we present a comprehensive mutagenesis based study of a region of the RSV F2 subunit (amino acids 75 - 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the pre-fusion form.

  • Characterization of an influenza virus pseudotyped with Ebolavirus glycoprotein [PublishAheadOfPrint]

  • We have produced a new Ebola virus pseudotype: E-S-FLU, which can be handled in biosafety level-1/2 containment for laboratory analysis. E-S-FLU is a single cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza haemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU production. Infection of cells by E-S-FLU was dependent on Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. E-S-FLU was neutralized specifically by anti-Ebola glycoprotein antibody and a variety of small drug molecules that are known to inhibit entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPACrreg;1280 Sigma) of 1280 pharmacologically active compounds for inhibition of virus entry. 215 compounds inhibited E-S-FLU infection, while only 22 inhibited the control H5-S-FLU virus coated in an H5 haemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action e.g. calcium channel blockers, estrogen receptor antagonists, anti-histamines, serotonin uptake inhibitors etc. and this correlates with inhibitor screening results with other pseudotypes or wild-type Ebola virus in the literature. E-S-FLU is a new tool for Ebola virus cell entry studies and is easily applied to high throughput screening assays for small molecule inhibitors or antibodies.

    Importance Ebola virus is from the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates of Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus, and screen for inhibitors. Here we characterized a new surrogate named E-S-FLU, that is based on a disabled influenza virus core coated with the Ebola virus surface protein, but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU in biosafety level-1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small molecule library of 1280 candidates for inhibitors of viral entry.

  • Inflammasome antagonism by human parainfluenza virus type 3 C protein [PublishAheadOfPrint]

  • Human parainfluenza virus type 3 (HPIV3) is a negative-sense single-stranded RNA virus belonging to the paramyxoviridae family. HPIV3 is a lung tropic virus causing airway diseases including pneumonia, croup, and bronchiolitis during infancy and childhood. Activation of inflammasome by pathogens results in production of pro-inflammatory cytokines like IL-1bbeta; during infection. Thus, inflammasome mediated pro-inflammatory response plays a critical role in regulating immune response and virus clearance. Inflammasome is a multimeric protein complex triggering caspase-1 activation. Activated caspase-1 cleaves pro-IL-1bbeta; to its mature (and active) secretory form. Our study revealed inflammasome activation in macrophages following HPIV3 infection. Specifically, activation of NLRP3/ASC inflammasome resulted in production of mature IL-1bbeta; from HPIV3 infected cells. Furthermore, TLR2 activation (first signal) and potassium efflux (second signal) constituted two cellular events mediating inflammasome activation following HPIV3 infection. During our studies, we surprisingly identified HPIV3 C protein as an antagonist of inflammasome activation. HPIV3 C protein is an accessory protein encoded by the open reading frame of viral phosphoprotein (P) gene. HPIV3 C protein interacted with NLRP3 protein and blocked inflammasome activation by promoting proteasomal degradation of NLRP3 protein. Thus, our studies report NLRP3/ASC inflammasome activation by HPIV3 via TLR2 signaling and potassium efflux. Furthermore, we have identified HPIV3 C as a viral component involved in antagonizing inflammasome activation.

    IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a paramyxovirus that causes respiratory tract diseases during infancy and childhood. Currently no effective vaccine or anti-viral therapy exists for HPIV3. Therefore, in order to develop anti-HPIV3 agents (therapeutics and vaccines) it is important to study HPIV3-host interaction during immune response. Inflammasome play an important role in immune response. Inflammasome activation by HPIV3 has not been previously reported. Our studies demonstrated inflammasome activation by HPIV3 in macrophages. Specifically, HPIV3 activated NLRP3/ASC inflammasome by TLR2 activation and potassium efflux. C proteins of paramyxoviruses are accessory proteins encoded by viral phosphoprotein gene. Role of C protein in inflammasome regulation was unknown. Surprisingly, our studies revealed that HPIV3 C protein antagonizes inflammasome activation. In addition, we highlighted for the first time a mechanism utilized by paramyxvirus accessory proteins to block inflammasome activation. HPIV3 C protein interacted with NLRP3 protein to trigger proteasomal degradation of NLRP3 protein.

  • Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence [PublishAheadOfPrint]

  • The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus, respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits (Oryctolagus cuniculus), that have recently been classified as GI strains. Yet, we could not explain the emergence of virulence since similar glycan specificities were found between several pathogenic and non-pathogenic strains. By contrast, EBHSV, recently classified as GII.1, bound to terminal bbeta;-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species (Oryctolagus cuniculus, Lepus europaeus and Sylvilagus floridanus) showed species-specific patterns regarding the susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagoviruses host specificity and range.

    IMPORTANCE Lagoviruses constitute a genus of the Caliciviridae family, comprising highly pathogenic viruses, RHDV and EBHSV, which infect rabbits and hares, respectively. Recently, non-pathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs are observed. The mechanisms responsible for the emergence of pathogenicity and host-species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely doors of virus entry. Here we studied the glycan-binding properties of novel pathogenic and non-pathogenic strains looking for a link between glycan-binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host range of the virus strains, suggesting that glycan diversity contributes to lagoviruses' host range.

  • Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (PRRSV) [PublishAheadOfPrint]

  • Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins, and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells are shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection is not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV.

    IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered composition confers numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naïve healthy cells. Furthermore, exosome-mediated viral transmission is largely resistant to PRRSV-specific neutralizing antibodies. Our study provides novel insights into an alternative mechanism of PRRSV transmission that can compromise the host's anti-PRRSV immune response.

  • Prasinovirus Attack of Ostreococcus Is Furtive by Day but Savage by Night [PublishAheadOfPrint]

  • Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life-cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analysed the life-cycle of OtV5, a lytic virus, using RNA-Seq from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 hours, but in the night viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the night appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4 % of the host's genes. Some host cells lysed progressively during the night, but a larger proportion lysed the following morning. Our data suggest that the life-cycles of algal viruses mirror the diurnal rhythms of their hosts.

    Importance Prasinoviruses are common in marine environments, and although several complete genomes of them and their hosts have been characterised, little is known about their life-cycles. Here we analyse in detail the transcriptional changes occurring over a 27 hour long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night, and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime when most energy (from light) is available, but during the night viral transcription takes off, accompanied by a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

  • Genetically intact but functionally impaired HIV-1 Env glycoproteins in the T-cell reservoir [PublishAheadOfPrint]

  • HIV-infected subjects under ART harbor a persistent viral reservoir in resting CD4+ T-cells, which accounts for the resurgence of HIV replication after ART interruption. A large majority of HIV reservoir genomes are genetically defective, but even among intact proviruses, few seem able to generate infectious virus. To understand this phenomenon, we have examined the function and expression of HIV envelope glycoproteins reactivated from the reservoir of 4 HIV-infected subjects under suppressive ART. We studied full-length genetically intact env sequences from both replicative viruses and cell-associated mRNAs. We found that these Env proteins varied extensively in fusogenicity and infectivity, with strongest functional defects found in Envs from cell-associated mRNAs. Env functional impairements were essentially explained by defects in Env protein expression. Our results support the idea that defects in HIV Env expression, preventing cytopathic or immune HIV clearance, contribute to the persistence of the HIV T-cell reservoir in vivo.

    Scientific importance In most individuals, evolution of HIV infection is efficiently controlled on the long-term by combination antiviral therapies. These treatments, however, fail to eradicate HIV from the infected subjects, a failure that results both in resurgence of virus replication and in resumption of HIV pathogenicity when the treatment is stopped. HIV resurgence, in these instances, is widely assumed to emerge from a reservoir of silent virus integrated in the genome of a small number of T lymphocytes. The silent HIV reservoir is mostly composed of heavily deleted or mutated HIV DNA. Moreover, among the seemingly intact remaining HIV, only very few are actually able to efficiently propagate in tissue culture. In this study, we find that intact HIV in the reservoir often carry strong defects in their capacity to promote fusion to neighboring cells and infection of target cells, a defect related to the function and expression of the HIV envelope glycoprotein. Impaired envelope glycoprotein expression and function could explain why cells harboring these viruses tend to remain undetected and unharmed in the reservoir.

  • Type III Interferon Restriction by Porcine Epidemic Diarrhea Virus and the Role of Viral Protein nsp1 in IRF1 Signaling [PublishAheadOfPrint]

  • Type III interferons (IFN-s) play a vital role to maintain the antiviral state of the mucosal epithelial surface in the gut, and in turn, enteric viruses may have evolved to evade the type III IFN responses during infection. To study of the possible immune evasion of porcine epidemic diarrhea virus (PEDV) from type III IFN response, a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. IFN-1 and IFN-3 inhibited the PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, ORF3, E, M, and N were found to suppress the type III IFN activities, and the IRF1 signaling mediated the suppression. PEDV specifically inhibited IRF1 nuclear translocation. Peroxisome is the innate antiviral signaling platform for activation of IRF1-mediated IFN- production, and peroxisomes were found to decrease in number in PEDV-infected cells. PEDV nsp1 blocked the nuclear translocation of IRF1 and reduced the number of peroxisomes to suppress IRF1-mediated type III IFNs. Mutational studies showed the conserved residues of nsp1 were crucial for IRF1-mediated IFN- suppression. Our study for the first time provides the evidence that the porcine enteric virus PEDV downregulates and evades the IRF1-mediated type III IFN responses by reducing the peroxisomes.

    IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus emerged in swine in the US and has caused severe economic losses. PEDV targets the intestinal epithelial cells in the gut, and intestinal epithelial cells selectively induce and respond to the production of type III interferons (IFNs). However, little is known about modulation of type III IFN response by PEDV in the intestinal epithelial cells. In this study, we established a porcine intestinal epithelial cell model for PEDV replication. We found that PEDV inhibited the IRF1-mediated type III IFN production by decreasing the peroxisomes in number in the porcine intestinal epithelial cells. We also demonstrated that the conserved residues in the PEDV nsp1 protein were crucial for IFN suppression. This study for the first time showed the PEDV evasion of type III IFN response in the intestinal epithelial cells. It provides valuable information on the host cell-virus interactions not only for PEDV but also other enteric viral infections in swine.

  • The absence of DHHC3 affects primary and latent HSV-1 infection [PublishAheadOfPrint]

  • UL20, an essential HSV-1 protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein {GODZ}). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in reduced palmitoylation of UL20 and altered localization, expression of UL20 and gK; the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps, and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1 infected GODZ-/- mice was significantly lower than in HSV-1 infected WT mice. The levels of UL20, gK and gB transcripts in the corneas of HSV-1 infected GODZ-/- mice on day 5 post infection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency/reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis is critically dependent on GODZ-mediated palmitoylation of viral UL20.

    IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70-90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection as well as reactivation in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

  • A proteomic survey of Junin virus interactions with human proteins reveals host factors required for arenavirus replication [PublishAheadOfPrint]

  • Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Juniiacute;n mammarenvirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Further, there is a little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside of cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid #1 particles. Bioinformatic analyses revealed that multiple classes of human proteins were overrepresented in the datasets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ARF1, ATP6V0D1 and PRDX3), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP5B, IMPDH2). Further, we show that release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This dataset provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.

    IMPORTANCE Arenaviruses are deadly human pathogens for which there are no United States Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Juniiacute;n mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a dataset that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

  • A general model for retroviral capsid pattern recognition by TRIM5 proteins [PublishAheadOfPrint]

  • Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5aalpha; uses a SPRY domain to bind capsids with low intrinsic affinity (KD ggt;1 mM), and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher (KD ~10 mmu;M) affinity for isolated capsid subunits. It has therefore been argued that TRIMCyp proteins may have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5aalpha;, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.

    IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5aalpha; protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5aalpha; lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5aalpha; SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5aalpha; RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5aalpha;, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits more tightly through its cyclophilin A domain, and was therefore thought to act independent of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5aalpha; and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.

  • Pathogenesis, humoral immune responses and transmission between co-housed animals in a ferret model of human RSV infection. [PublishAheadOfPrint]

  • Small animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Amongst those described to date, infections in cotton rats, mice, guinea pigs, chinchillas and Syrian hamsters with hRSV strains Long and/or A2 have been well characterised, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection but the pathogenesis and immune responses elicited following infection have not been well characterised. Herein, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterised virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0-15 post-infection) were similar between the two strains and both elicited high levels of F glycoprotein-specific binding and neutralising antibodies following virus clearance (days 16-22 post-infection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to co-housed naïve recipients and have characterised virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV.

    IMPORTANCE Ferrets have been widely used to study pathogenesis, immunity and transmission following human influenza virus infections, however far less is known regarding the utility of the ferret model to study hRSV infections. Following intranasal (IN) infection of adult ferrets with the well characterised Long or A2 strains of hRSV, we report virus replication and cytokine induction in the upper and lower airways, as well as the development of virus-specific humoral responses. Importantly, we demonstrate transmission of hRSV from experimentally infected donor ferrets to co-housed naïve recipients. Together, these findings significantly enhance our understanding of the utility of the ferret as a small animal model to investigate aspects of hRSV pathogenesis and immunity.

  • Novel insect-specific Eilat virus-based chimeric vaccine candidates provide durable, mono- and multi-valent, single dose protection against lethal alphavirus challenge [PublishAheadOfPrint]

  • Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals. EILV chimeras containing structural proteins of EEEV or VEEV were engineered and successfully rescued in Aedes albopictus cells. Cryo-EM reconstructions at 8 and 11 AAring; of EILV/VEEV and EILV/EEEV, respectively, showed virion and glycoprotein spike structures similar to VEEV-Tc83 and other alphaviruses. The chimeras were unable to replicate in vertebrate cell lines or in brains of newborn mice when injected intracranially. Histopathologic examinations of the brain tissues showed no evidence of pathologic lesions, and were indistinguishable from those of mock-infected animals. A single-dose immunization of either monovalent or multivalent EILV chimera(s) generated neutralizing antibody responses and protected animals against lethal challenge 70 days later. Lastly, a single dose of monovalent EILV chimeras generated protective responses as early as day 1 post-vaccination, and partial or complete protection by day 6. These data demonstrate the safety, immunogenicity, and efficacy of novel insect-specific EILV-based chimeras as potential EEEV and VEEV vaccines.

    IMPORTANCE Mostly in the last decade, insect-specific viruses have been discovered in several arbovirus families. However, most of these viruses are not well studied and largely have been ignored. We explored use of the mosquito-specific alphavirus, Eilat virus (EILV), as an alphavirus vaccine platform in well-established disease models for eastern (EEE) and Venezuelan equine encephalitis (VEE). EILV-based chimeras replicated to high titers in a mosquito cell line, yet retained their host range restriction in vertebrates both in vitro and in vivo. In addition, the chimeras generated immune responses that were higher than other human and/or equine vaccines. These findings indicate the feasibility of producing a safe, efficacious, mono- or multi-valent vaccine against the encephalitic alphaviruses, VEEV and EEEV. Lastly, these data demonstrate how host restricted, insect-specific viruses can be engineered to develop vaccines against related pathogenic arboviruses that cause severe disease in humans and domesticated animals.

  • Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein is Crucial for Production of Infectious Virus Particles [PublishAheadOfPrint]

  • Virus replication is mediated by interactions between virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78-ggt;Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in production of infectious virus particles. M2-K78R mutant progeny contained more HA proteins, less viral RNAs and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination under electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with M1 protein to a lesser extent than for wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal level of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that M2-K78R mutant virus induced autophagy and apoptosis earlier than wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating efficient packaging of the viral genome into virus particles and timing of viral-induced cell death.

    IMPORTANCE Annual epidemics and recurring pandemics of influenza viruses represent a very high global health and economic burden. Influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in viral entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N-terminus of M2 proteins. However, viruses resistant to Rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

  • Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1 [PublishAheadOfPrint]

  • Many RNA viruses exist as an ensemble of genetically-diverse, replicating populations, known as mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds of varying genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 (ORF1) or of the full-genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency of the order of 10-3 per nucleotide copied, and existence of marked genetic diversity with a high normalized Shannon entropy. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of varying sizes and compositions on phenotypic characteristics such as the replication level of (+)-RNA progeny, amount of double-stranded RNA (a surrogate for rate of viral replication) and ORF1 protein, and expression of interferon stimulating genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help provide further insights into virus evolution and prediction of emerging viral diseases.


    Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro. We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a ss(+)RNA virus, with varying and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effect of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud, which had an extremely highgenetic diversity, had replication rate exceeding that of the wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

  • Glycine-zipper motifs in hepatitis C virus nonstructural protein 4B are required for the establishment of viral replication organelles [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single, double and multi membrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms how this protein contributes to organelle biogenesis. Using forward and reverse genetics we identified glycine-zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine-zippers in NS4B homo and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine-zipper residues for the subcellular distribution and the morphology of HCV-induced double membrane vesicles. Notably, loss-of-function NS4B glycine-zipper mutants prominently induced single membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double membrane vesicle formation. These findings highlight a so far unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.

    IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of hepatitis C virus (HCV), expression of the nonstructural proteins induces the accumulation of double membrane vesicles that likely arise from a concerted action of viral and co-opted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine-zipper motifs within HCV nonstructural protein 4B (NS4B) transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and thus, efficient viral RNA replication. These results reveal how glycine-zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and thus, viral RNA replication.

  • Identification and functional characterization of phosphorylation sites of the HPV31 E8^E2 protein [PublishAheadOfPrint]

  • The papillomavirus E2 protein regulates transcription, replication and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA-binding activity and chromosomal attachment. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of HPV31 E8^E2 and E2 proteins identify phosphorylated S78, S81 and S100 in E8^E2 and S266 and S269 in E2 in their hinge regions. Phos-Tag analyses of wildtype and mutant proteins indicate that S78 is a major phosphorylation site in E8^E2 but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8^E2rrsquo; s repression activity of reporter constructs whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8^E2rrsquo; s protein stability, nuclear localization, binding to DNA or to cellular NCoR/SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8^E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho-S78 E8^E2 dependent manner. In summary, our data suggest that phosphorylation of S78 in E8^E2 regulates its repression activity by a novel mechanism and this seems to be important for the modulation of host cell gene expression but not viral replication.


    Post-translational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of Serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8^E2 is phosphorylated at S78 in the hinge region and this is important for E8^E2rrsquo; s repression activity. Surprisingly, phosphorylation at S78 in E8^E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.


  • The well-characterised association between HLA-B*27:05 and protection against HIV disease progression has been linked to immunodominant HLA-B*27:05-restricted CD8+ T-cell responses towards the conserved Gag 263-272 (llsquo;KK10rrsquo;) and Pol 901-909 llsquo;KY9rrsquo; epitopes. We here studied the impact of the 3 amino acid differences between HLA-B*27:05 and the closely-related HLA-B*27:02 on the HIV-specific CD8+ T-cell response hierarchy and on immune control of HIV. Genetic epidemiological data indicate that both HLA-B*27:02 and HLA-B*27:05 associate with slower disease progression and lower viral loads. The effect of HLA-B*27:02 appears consistently stronger than that of HLA-B*27:05. In contrast to HLA-B*27:05, the immunodominant HIV-specific HLA-B*27:02-restricted CD8+ T-cell response is to a Nef epitope (residues 142-150, llsquo;VW9rrsquo;), with Pol-KY9 subdominant and Gag-KK10 further subdominant. This selection was driven by structural differences in the F-pocket, mediated by a polymorphism between these two HLA alleles at position 81. Analysis of autologous virus sequences showed that in HLA-B*27:02-positive subjects all three of these CD8+ T-cell responses impose selection pressure on the virus, whereas in HLA-B*27:05-positive subjects there is no Nef-VW9-mediated selection pressure. These studies demonstrate that HLA-B*27:02 mediates protection against HIV disease progression that is at least as strong or stronger than that mediated by HLA-B*27:05. In combination with the protective Gag-KK10 and Pol-KY9 CD8+ T-cell responses that dominate HIV-specific CD8+ T-cell activity in HLA-B*27:05-positive subjects, a Nef-VW9-specific response is additionally present and immunodominant in HLA-B*27:02-positive subjects, mediated through a polymorphism at residue 81 in the F-pocket, that contributes to selection pressure against HIV.

    IMPORTANCE CD8+ T-cells play a central role in successful control of HIV infection, and have the potential also to mediate the eradication of viral reservoirs of infection. The principal means by which llsquo;protectiverrsquo; HLA class I molecules, such as HLA-B*27:05 and HLA-B*57:01, slow HIV disease progression, is believed to be via the particular HIV-specific CD8+ T cell responses restricted by those alleles. We focus here on HLA-B*27:05, one of the best-characterised llsquo;protectiverrsquo; HLA molecules, and the closely-related HLA-B*27:02, which differs by only 3 amino acids, and which has not been well-studied in relation to control of HIV infection. We show that HLA-B*27:02 is also protective against HIV disease progression, but the CD8+ T-cell immunodominance hierarchy of HLA-B*27:02 differs strikingly from that of HLA-B*27:05. These findings indicate that the immunodominant HLA-B*27:02-restricted Nef response adds to protection mediated by the Gag and Pol specificities that dominate anti-HIV CD8+ T-cell activity in HLA-B*27:05-positive subjects.

  • Merkel cell polyomavirus infection of animal dermal fibroblasts [PublishAheadOfPrint]

  • Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the infectability of MCPyV-GFP pseudovirus, MCPyV recombinant virion, and several MCPyV chimeric viruses in dermal fibroblasts isolated from various model animals, including mouse (Mus musculus), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), patas monkey (Erythrocebus patas), common woolly monkey (Lagothrix lagotricha), red-chested mustached tamarin (Saguinus labiatus), and tree shrew (Tupaia Belangeri). We found that MCPyV-GFP pseudovirus is able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus, in which the entire enhancer region of MCPyV early promoter has been replaced with the SV40 analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying the viral oncogene function during MCC oncogenic progression.

    IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel cell carcinoma (MCC). With the increasing number of MCC diagnoses, there is a need to better understand the virus and its oncogenic potential. However, studies attempting to fully elucidate MCPyV oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. To pinpoint the best candidate for developing an MCPyV infection animal model, we examined the MCPyV infectability of dermal fibroblasts isolated from various established model animals. Of the animal cell types we tested, chimpanzee dermal fibroblasts were the only isolates that support the full MCPyV infectious cycle. To overcome the infection blockade in the other model animals, we constructed chimeric viruses that achieved robust MCPyV entry and oncogene expression in rat fibroblasts. Our results suggest that the rat may serve as an in vivo model to study MCV oncogenesis.

  • Comparison of the Efficacy of N9 Neuraminidase-specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection [PublishAheadOfPrint]

  • The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses and emergence of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be an important contributor to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. Two major antigenic regions, i.e., the 250-loop and 370/400/430-loop domains, were identified. MAbs 1E8, 2F6, 10F4 and 11B2, which recognize these 2 antigenic domains, were characterized in depth. These 4 MAbs differ in ability to inhibit cleavage of small and large substrates (MU-NANA and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All 4 MAbs reduced the in vitro spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations, but to different degrees. These MAbs have different in vivo effectiveness, 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection, and suggests that in vitro properties can be used to rank antibodies with therapeutic potential.

    IMPORTANCE The novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses, some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies in vitro measures that can be used to predict the in vivo efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.

  • Antibodies directed towards neuraminidase N1 control disease in a mouse model of influenza [PublishAheadOfPrint]

  • There is increasing evidence to suggest that antibodies directed towards influenza A virus (IAV) neuraminidase (NA) are an important correlate of protection against influenza in humans. Moreover, the potential of NA-specific antibodies to provide broader protection than conventional hemagglutinin (HA) antibodies has been recognized. Herein, we describe the isolation of two monoclonal antibodies, N1-7D3 and N1-C4, directed towards the N1 NA. N1-7D3 binds to a conserved linear epitope in the membrane distal, carboxy-terminal part of the NA and reacted with the NA of seasonal H1N1 isolates ranging from 1977 till 2007 the 2009 H1N1pdm virus as well as A/Vietnam/1194/04 (H5N1). However, N1-7D3 lacked NA inhibition (NI) activity and the ability to protect BALB/c mice against a lethal challenge with a range of H1N1 viruses. Conversely, N1-C4 bound to a conformational epitope that is conserved between two influenza subtypes, the 2009 H1N1pdm and H5N1 IAV and displayed potent in vitro antiviral activity mediating both NI and plaque size-reduction. Moreover, N1-C4 could provide heterosubtypic protection in BALB/c mice against a lethal challenge with 2009 H1N1pdm or H5N1 virus. Glutamic acid residue 311 in the NA was found to be critical for the NA binding and antiviral activity of monoclonal antibody N1-C4. Our data provide further evidence on cross-protective epitopes within the N1 subtype and highlight the potential of NA as an important target for vaccine and therapeutic approaches.

    Importance Influenza remains a world-wide burden to public health. As such the development of new and novel vaccines and therapeutics against influenza virus is crucial. Human challenge studies have recently highlighted the importance of antibodies directed towards the viral neuraminidase (NA) as an important correlate of reduced influenza-associated disease severity. Furthermore, there is evidence that anti-NA antibodies can provide broader protection than antibodies towards the viral hemagglutinin. Here we describe the isolation and detailed characterization of two N1 NA-specific monoclonal antibodies. One of these monoclonal antibodies broadly binds N1 type NAs and the second one displays NAI, in vitro and in vivo anti-viral activity against 2009 H1N1pdm and H5N1 influenza viruses. These two new anti-NA antibodies contribute to our understanding of the antigenic properties and protective potential of the influenza NA antigen.

  • Inhibition of cytosolic phospholipase A2{alpha} impairs an early step of coronavirus replication in cell culture [PublishAheadOfPrint]

  • Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double membrane vesicles (DMV) and other membranous structures that are referred to as replicative organelles (RO). The latter provide a structural scaffold for viral replication/transcription complexes (RTC) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand (+) RNA virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2aalpha; (cPLA2aalpha;) in coronavirus replication using a small-molecular-weight non-peptidic inhibitor (Py-2). Inhibition of cPLA2aalpha; activity, which produces lysophospholipids (LPL) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells and (iii) this increase was diminished in the presence of cPLA2aalpha; inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2aalpha; activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.


    Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2aalpha;, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments at which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2aalpha; inhibitor.

  • HAdV protein V core protein is targeted by the host SUMOylation machinery to limit essential viral functions [PublishAheadOfPrint]

  • Human Adenoviruses (HAdV) are non-enveloped containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear-pore-complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood.

    The core protein V is speculated to bridge core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultanously, these altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than observed during wildtype infection.

    Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies.


    Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion, as the functions are more diverse. We showed that capsid protein protein VI is regulating antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, being beneficial for efficient viral gene expression. Here, we were able to show that also core protein V represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. Conclusively, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency.

  • An Insight Into The Role of E3 ubiquitin Ligase c-Cbl, ESCRT Machinery and Host Cell Signaling in Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Entry and Trafficking [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro infection of dermal endothelial cell begins with its binding to host cell surface receptor molecules such as heparan sulfate (HS), integrins (aalpha;3bbeta;1, aalpha;Vbbeta;3 and aalpha;Vbbeta;5), xCT and EphA2 receptor tyrosine kinase (EphA2R). These initial events initiate dynamic host protein-protein interactions involving a multi-molecular complex of receptors, signal molecules (FAK, Src, PI3-K, RhoA-GTPase), adaptors (c-Cbl, CIB1, Crk, p130Cas and GEF-C3G), and actin/myosin II light chain that leads to virus entry via macropinocytosis. Here we discuss how KSHV hijacks c-Cbl, an E3 ubiquitin ligase, to monoubiquitinate the receptors/actin which acts like markers for trafficking (similar to zip codes), resulting in the recruitment of the members of the host endosomal sorting complexes required for transport (ESCRT) Hrs, Tsg101, EAP45, CHMP 5 and 6 proteins (zip code readers) recognizing the ubiquitinated proteins and adaptors machinery to traffic through the different endosomal compartments in the cytoplasm to initiate the macropinocytic process and infection.

  • A novel role of vBcl2 in the virion assembly of Kaposi's sarcoma-associated herpesvirus [PublishAheadOfPrint]

  • The viral Bcl-2 homolog (vBcl2) of Kaposi's sarcoma-associated herpesvirus (KSHV) displays efficient anti-apoptotic and anti-autophagic activity through its central BH3 domain, which functions to prolong the lifespan of virus-infected cells and ultimately enhances viral replication and latency. Independent on its anti-apoptotic and anti-autophagic activity, vBcl2 also plays an essential role in KSHV lytic replication through its amino-terminal 11-20 amino acids (aa). Here, we report a novel molecular mechanism of vBcl2-mediated regulation of KSHV lytic replication. vBcl2 specifically bound the tegument protein ORF55 through its amino-terminal 11-20aa, allowing their association with virions. Consequently, the vBcl2p peptide derived from the vBcl2 11-20aa effectively disrupted the interaction between vBcl2 and ORF55, inhibiting the incorporation of ORF55 tegument protein into virions. This study provides new insight of vBcl2's function in KSHV virion assembly that is separable from its inhibitory role of host apoptosis and autophagy.

    IMOPRTANCE KSHV, an important human pathogen accounting for a large percentage of virally caused cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish a life-long persistent infection. Upon viral infection, infected cells can go through a programmed cell death, including apoptosis and autophagy, that plays an effective role in anti-viral responses. To counter host response, the KSHV vBcl2 efficiently blocks apoptosis and autophagy to persist the lifespan of virus-infected cells. Besides its anti-programmed cell death activity, vBcl2 also interacts with ORF55 tegument protein for virion assembly in infected cells. Interestingly, the vBcl2p peptide disrupts the vBcl2-ORF55 interaction and effectively inhibits KSHV virion assembly. This study indicates that the KSHV vBcl2 harbors at least three genetically separable functions to modulate both host cell death signaling and virion production, and that the vBcl2p peptide can be developed as an anti-KSHV therapeutic application.

  • Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections [PublishAheadOfPrint]

  • Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 AAring; resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.

    IMPORTANCE Coronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease-resistant which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts.

  • Cryo-EM structure of porcine delta coronavirus spike protein in the pre-fusion state [PublishAheadOfPrint]

  • Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here we determined the cryo-EM structure of porcine delta coronavirus (PdCoV) spike protein at 3.3-angstrom resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane-fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and beta-coronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alpha-coronavirus S1-CTDs, but its structure differs from that of beta-coronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the pre-fusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that delta-coronavirus spikes are structurally and evolutionally more closely related to alpha-coronavirus spikes than to beta-coronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by delta-coronaviruses as well as coronaviruses in general.

    SIGNIFICANCE In this study we determined the cryo-EM structure of porcine delta coronavirus (PdCoV) spike protein at 3.3 angstrom. This is the first atomic structure of a spike protein from the delta coronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and beta-coronavirus spike proteins. In the current study, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of coronavirus spike proteins, and provides many insights into the receptor recognition, membrane fusion, immune evasion, and evolution of PdCoV spike protein.

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