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

Table of Contents for this page:

  • Current Issue
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  • Current Issue of Journal of Virology

    JVI Current Issue

  • Unique Structural Features of Influenza Virus H15 Hemagglutinin [Virus-Cell Interactions]

  • Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.

    IMPORTANCE In the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.

  • Development of an Alternative Modified Live Influenza B Virus Vaccine [Vaccines and Antiviral Agents]

  • Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (ts) mutations into the PB1 and PB2 subunits along with a modified HA epitope tag in the C terminus of PB1 resulted in influenza A viruses (IAV) that are safe and effective as modified live attenuated (att) virus vaccines (IAV att). We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo. Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines.

    IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons (2013nndash;2014, 2014nndash;2015, and 2015nndash;2016). While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an alternative strategy, the incorporation of 2 amino acid mutations and a modified HA tag at the C terminus of PB1, which is sufficient to attenuate the IBV. As a LAIV, this novel vaccine provides complete protection against IBV strains. The availability of attenuated IAV and IBV backbones based on contemporary strains offers alternative platforms for the development of LAIVs that may overcome current limitations.

  • An Immortalized Human Dorsal Root Ganglion Cell Line Provides a Novel Context To Study Herpes Simplex Virus 1 Latency and Reactivation [Genome Replication and Regulation of Viral Gene Expression]

  • A defining characteristic of alphaherpesviruses is the establishment of lifelong latency in host sensory ganglia with occasional reactivation causing recurrent lytic infections. As an alternative to rodent models, we explored the use of an immortalized cell line derived from human dorsal root ganglia. HD10.6 cells proliferate by virtue of a transduced tetracycline-regulated v-myc oncogene. In the presence of doxycycline, HD10.6 cells mature to exhibit neuronal morphology and express sensory neuron-associated markers such as neurotrophin receptors TrkA, TrkB, TrkC, and RET and the sensory neurofilament peripherin. Infection of mature HD10.6 neurons by herpes simplex virus 1 (HSV-1) results in a delayed but productive infection. However, infection at a low multiplicity of infection (MOI) in the presence of acyclovir results in a quiescent infection resembling latency in which viral genomes are retained in a low number of neurons, viral gene expression is minimal, and infectious virus is not released. At least some of the quiescent viral genomes retain the capacity to reactivate, resulting in viral DNA replication and release of infectious virus. Reactivation can be induced by depletion of nerve growth factor; other commonly used reactivation stimuli have no significant effect.

    IMPORTANCE Infections by herpes simplex viruses (HSV) cause painful cold sores or genital lesions in many people; less often, they affect the eye or even the brain. After the initial infection, the virus remains inactive or latent in nerve cells that sense the region where that infection occurred. To learn how virus maintains and reactivates from latency, studies are done in neurons taken from rodents or in whole animals to preserve the full context of infection. However, some cellular mechanisms involved in HSV infection in rodents are different from those in humans. We describe the use of a human cell line that has the properties of a sensory neuron. HSV infection in these cultured cells shows the properties expected for a latent infection, including reactivation to produce newly infectious virus. Thus, we now have a cell culture model for latency that is derived from the normal host for this virus.

  • Editorial Board [Masthead]

  • Vertex-Specific Proteins pUL17 and pUL25 Mechanically Reinforce Herpes Simplex Virus Capsids [Structure and Assembly]

  • Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of icosahedral herpes simplex virus capsids. pUL17 and pUL25, which form the capsid vertex-specific component (CVSC), particularly contributed to capsid resilience along the 5-fold and 2-fold but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry of the protein composition of the capsids, revealed that both pUL17 and pUL25 are required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids.

    IMPORTANCE In this study, the structural, material properties of herpes simplex virus 1 were investigated. The capsid of herpes simplex virus is built up of a variety of proteins, and we scrutinized the influence of two of these proteins on the stability of the capsid. For this, we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach, we revealed that both studied proteins play an essential role in viral stability. These new insights support us in forming a complete view on viral structure and furthermore could possibly help not only to develop specific antivirals but also to build protein shells with improved stability for drug delivery purposes.

  • ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells [Virus-Cell Interactions]

  • Although a varicella-zoster virus (VZV) vaccine has been used for many years, the neuropathy caused by VZV infection is still a major health concern. Open reading frame 7 (ORF7) of VZV has been recognized as a neurotropic gene in vivo, but its neurovirulent role remains unclear. In the present study, we investigated the effect of ORF7 deletion on VZV replication cycle at virus entry, genome replication, gene expression, capsid assembly and cytoplasmic envelopment, and transcellular transmission in differentiated neural progenitor cells (dNPCs) and neuroblastoma SH-SY5Y (dSY5Y) cells. Our results demonstrate that the ORF7 protein is a component of the tegument layer of VZV virions. Deleting ORF7 did not affect viral entry, viral genome replication, or the expression of typical viral genes but clearly impacted cytoplasmic envelopment of VZV capsids, resulting in a dramatic increase of envelope-defective particles and a decrease in intact virions. The defect was more severe in differentiated neuronal cells of dNPCs and dSY5Y. ORF7 deletion also impaired transmission of ORF7-deficient virus among the neuronal cells. These results indicate that ORF7 is required for cytoplasmic envelopment of VZV capsids, virus transmission among neuronal cells, and probably the neuropathy induced by VZV infection.

    IMPORTANCE The neurological damage caused by varicella-zoster virus (VZV) reactivation is commonly manifested as clinical problems. Thus, identifying viral neurovirulent genes and characterizing their functions are important for relieving VZV related neurological complications. ORF7 has been previously identified as a potential neurotropic gene, but its involvement in VZV replication is unclear. In this study, we found that ORF7 is required for VZV cytoplasmic envelopment in differentiated neuronal cells, and the envelopment deficiency caused by ORF7 deletion results in poor dissemination of VZV among neuronal cells. These findings imply that ORF7 plays a role in neuropathy, highlighting a potential strategy to develop a neurovirulence-attenuated vaccine against chickenpox and herpes zoster and providing a new target for intervention of neuropathy induced by VZV.

  • Molecular Evolution and Emergence of H5N6 Avian Influenza Virus in Central China [Genetic Diversity and Evolution]

  • H5N6 avian influenza virus (AIV) has posed a potential threat to public health since its emergence in China in 2013. To understand the evolution and emergence of H5N6 AIV in the avian population, we performed molecular surveillance of live poultry markets (LPMs) in Wugang Prefecture, Hunan Province, in central China, during 2014 and 2015. Wugang Prefecture is located on the Eastern Asian-Australian migratory bird flyway, and a human death due to an H5N6 virus was reported in the prefecture on 21 November 2016. In total, we sampled and sequenced the complete genomes of 175 H5N6 AIVs. Notably, our analysis revealed that H5N6 AIVs contain at least six genotypes arising from segment reassortment, including a rare variant that possesses an HA gene derived from H5N1 clade 2.3.2 and a novel NP gene that has its origins with H7N3 viruses. In addition, phylogenetic analysis revealed that genetically similar H5N6 AIVs tend to cluster according to their geographic regions of origin. These results help to reveal the evolutionary behavior of influenza viruses prior to their emergence in humans.

    IMPORTANCE The newly emerged H5N6 influenza A virus has caused more than 10 human deaths in China since 2013. In November 2016, a human death due to an H5N6 virus, in Wugang Prefecture, Hunan Province, was confirmed by the WHO. To better understand the evolution and emergence of H5N6 viruses, we surveyed live poultry markets (LPMs) in Wugang Prefecture before the reported human death, with a focus on revealing the diversity and genomic origins of H5N6 in birds during 2014 and 2015. In general, H5N6 viruses in this region were most closely related to H5N1 clade 2.3.4.4, with the exception of one virus with an HA gene derived from clade 2.3.2 such that it represents a novel reassortant. Clearly, the ongoing surveillance of LPMs is central to monitoring the emergence of pathogenic influenza viruses.

  • MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2 [Genome Replication and Regulation of Viral Gene Expression]

  • Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via various mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection.

    IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.

  • Overactive Epidermal Growth Factor Receptor Signaling Leads to Increased Fibrosis after Severe Acute Respiratory Syndrome Coronavirus Infection [Pathogenesis and Immunity]

  • Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic respiratory virus that causes morbidity and mortality in humans. After infection with SARS-CoV, the acute lung injury caused by the virus must be repaired to regain lung function. A dysregulation in this wound healing process leads to fibrosis. Many survivors of SARS-CoV infection develop pulmonary fibrosis (PF), with higher prevalence in older patients. Using mouse models of SARS-CoV pathogenesis, we have identified that the wound repair pathway, controlled by the epidermal growth factor receptor (EGFR), is critical to recovery from SARS-CoV-induced tissue damage. In mice with constitutively active EGFR [EGFR(DSK5) mice], we find that SARS-CoV infection causes enhanced lung disease. Importantly, we show that during infection, the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) are upregulated, and exogenous addition of these ligands during infection leads to enhanced lung disease and altered wound healing dynamics. Our data demonstrate a key role of EGFR in the host response to SARS-CoV and how it may be implicated in lung disease induced by other highly pathogenic respiratory viruses.

    IMPORTANCE PF has many causative triggers, including severe respiratory viruses such as SARS-CoV. Currently there are no treatments to prevent the onset or limit the progression of PF, and the molecular pathways underlying the development of PF are not well understood. In this study, we identified a role for the balanced control of EGFR signaling as a key factor in progression to PF. These data demonstrate that therapeutic treatment modulating EGFR activation could protect against PF development caused by severe respiratory virus infection.

  • Homologous Capsid Proteins Testify to the Common Ancestry of Retroviruses, Caulimoviruses, Pseudoviruses, and Metaviruses [Letters to the Editor]

  • SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum [Virus-Cell Interactions]

  • Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.

    IMPORTANCE How a nonenveloped virus transports across a biological membrane to cause infection remains mysterious. One enigmatic step is whether host cytosolic components are co-opted to transport the viral particle into the cytosol. During ER-to-cytosol membrane transport of the nonenveloped polyomavirus SV40, a decisive infection step, a cytosolic complex composed of Hsc70-SGTA-Hsp105 was previously shown to associate with the ER membrane. SGTA and Hsp105 have been shown to extract SV40 from the ER and transport the virus into the cytosol. We demonstrate here a critical role of Hsc70 in SV40 ER-to-cytosol penetration and reveal how SGTA controls Hsc70 to impact this process.

  • Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing [Genome Replication and Regulation of Viral Gene Expression]

  • Parvoviruses use a variety of means to control the expression of their compact genomes. The bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host cell-dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing.

    IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner.

  • Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes [Vaccines and Antiviral Agents]

  • Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes.

    IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.

  • Filamentous Structures Induced by a Phytoreovirus Mediate Viral Release from Salivary Glands in Its Insect Vector [Virus-Cell Interactions]

  • Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae, induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis. Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis. Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis. For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors.

    IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.

  • Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes [Structure and Assembly]

  • Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (Naamp;B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by Naamp;B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.

    IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.

  • Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells [Virus-Cell Interactions]

  • Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM.

    IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

  • Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies [Pathogenesis and Immunity]

  • Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies.

    IMPORTANCE Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.

  • Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model [Vaccines and Antiviral Agents]

  • Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection.

    IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed.

  • Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly [Structure and Assembly]

  • The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.

    IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.

  • Potato Virus Y HCPro Suppression of Antiviral Silencing in Nicotiana benthamiana Plants Correlates with Its Ability To Bind In Vivo to 21- and 22-Nucleotide Small RNAs of Viral Sequence [Pathogenesis and Immunity]

  • We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21- and 22-nt vsRNAs. HCPro constituted at least 54% of the total protein content in purified preparations, and we were able to calculate its contribution to the 21- and the 22-nt pools of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21-nt vsRNAs of the HCPro preparation, 5'-terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes or of plant sequences.

    IMPORTANCE It was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs) in vitro and, in the case of Turnip mosaic virus HCPro, also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro binds in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression-of-silencing-deficient HCPro mutant that can accumulate in this host when expressed from a virus vector, we also show that sRNA binding correlates with silencing suppression activity. We demonstrate that HCPro binds at least to sRNAs with viral sequences of 21 nucleotides (nt) and, to a much lesser extent, of 22 nt, which were are also differentially enriched in 5'-end adenines relative to the purified controls. Together, our results support the physical binding of HCPro to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.

  • Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells [Pathogenesis and Immunity]

  • Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (llt;1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine.

    IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We studied CMV acquisition in infants and found that infections are usually brief and self-limited and are successfully established relatively rarely. Thus, although most people eventually acquire CMV infection, it usually requires numerous exposures. Our analyses indicate that this is because the virus is surprisingly inefficient, barely replicating well enough to spread to neighboring cells in the mouth. Greater knowledge of why CMV infection usually fails may provide insight into how to prevent it from succeeding.

  • Cbl E3 Ligase Mediates the Removal of Nectin-1 from the Surface of Herpes Simplex Virus 1-Infected Cells [Virus-Cell Interactions]

  • The Cbl E3 ligase has been linked to the down-modulation of surface signaling responses by inducing internalization of surface receptors. The adaptor protein CIN85 is a partner of Cbl that augments many of these interactions. Previously, an interaction was demonstrated between ICP0 and CIN85, which results in the removal of epidermal growth factor receptor (EGFR) from the surface of the infected cells with a concomitant attenuation of EGFR signaling. Here, we examined whether Cbl mediates the removal of the herpes simplex virus 1 (HSV-1) entry receptor Nectin-1 from the surface of infected cells. We found the following: (i) that Cbl, Nectin-1, and the viral glycoprotein D (gD) form a complex in infected cells; (ii) that during infection Nectin-1 is removed from the surface of the infected cells but is retained on the surface of cells that have been depleted of Cbl; and (iii) that in cells infected with a ICP0 mutant virus, Nectin-1 remained on the cell surface. Thus, Cbl is necessary but not sufficient for the removal of Nectin-1 from the cell surface. In addition, we observed that in Cbl-depleted cells there was enhanced entry after infection. These cells were susceptible to secondary infections by HSV-1. Viral entry in CIN85-depleted cells was only moderately enhanced compared to that in the Cbl-depleted cells, suggesting that the Cblnndash;Nectin-1 interaction is likely the key to the downregulation of surface Nectin-1. The removal of the HSV-1 entry receptor Nectin-1 from the surface of the infected cells may be part of the strategy of the virus to efficiently spread to uninfected cells.

    IMPORTANCE The Cbl E3 ligase suppresses surface signaling responses by inducing internalization of surface components. The targets of Cbl include such components as immune system receptors, growth factor receptors, adhesion, and cell-to-cell contact molecules. The immediate early protein ICP0 of herpes simplex virus 1 (HSV-1) interacts with CIN85, an adaptor protein that augments Cbl functions. The consequence of this interaction is the removal of the epidermal growth factor receptor (EGFR) from the surface of the infected cells with concomitant suppression of the EGF ligand signaling. The viral entry receptor Nectin-1 is also internalized during HSV-1 infection in a Cbl-dependent mechanism, and that increases the opportunity of the virus to spread to uninfected cells. The diversion of the Cbl/CIN85 endocytic machinery may be a strategy utilized by the virus to alter the cell surface pattern to prevent detrimental host responses.

  • Erratum for Purviance et al., "In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase {alpha}-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides" [Errata]

  • Classic Spotlight, 1990 and 1991: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • Understanding the Mechanism of the Broad-Spectrum Antiviral Activity of Favipiravir (T-705): Key Role of the F1 Motif of the Viral Polymerase [Vaccines and Antiviral Agents]

  • Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.

    IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.

  • Erratum for Nilsson et al., "Role of the PB2 627 Domain in Influenza A Virus Polymerase Function" [Errata]

  • Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection [Cellular Response to Infection]

  • Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae. During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-bbeta;) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo. Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

    IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo. Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

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

  • Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments [Genome Replication and Regulation of Viral Gene Expression]

  • The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMS1 to -8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explore the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1 to -4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, -7, -9, and -17 to -19) around pTMS1 and -2 displayed a novel phenotype showing a ggt;1,000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious-virus-like particle yields. HEK-293 cells infected with the six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal-mutant viruses revealed two consensus reversion mutations, leucine to phenylalanine at codon 181 (L181F) within pTMS7 of NS2A and isoleucine to threonine at codon 114 (I114T) within NS2B. The introduction of an NS2A-L181F mutation into the lethal (NM15, -16, -25, and -33) and CPE-defective (NM7, -9, and -19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas the NS2B-L114T mutation rescued the NM16, -25, and -33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between the NS2A and NS2B proteins were also implicated.

    IMPORTANCE The characterization of the N-terminal (current study) and C-terminal halves of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle. A novel region responsible for virus-induced cytopathic effect (CPE) within pTMS1 and -2 of DENV NS2A was identified. Revertant genetics studies implied unexpected relationships between various pTMSs of DENV NS2A and NS2B. These results provide comprehensive information regarding the functions of DENV NS2A and the specific amino acids and transmembrane segments responsible for these functions. The positions and properties of the rescuing mutations were also revealed, providing important clues regarding the manner in which intramolecular or intermolecular interactions between the pTMSs of NS2A and NS2B regulate virus replication, assembly/secretion, and virus-induced CPE. These results expand the understanding of flavivirus replication. The knowledge may also facilitate studies of pathogenesis and novel vaccine and antiflaviviral drug development.

  • Double Plant Homeodomain Fingers 2 (DPF2) Promotes the Immune Escape of Influenza Virus by Suppressing Beta Interferon Production [Cellular Response to Infection]

  • The high mutation rates of the influenza virus genome facilitate the generation of viral escape mutants, rendering vaccines and drugs against influenza virus-encoded targets potentially ineffective. Therefore, we identified host cell determinants dispensable for the host but crucial for virus replication, with the goal of preventing viral escape and finding effective antivirals. To identify these host factors, we screened 2,732 human genes using RNA interference and focused on one of the identified host factors, the double plant homeodomain fingers 2 (DPF2/REQ) gene, for this study. We found that knockdown of DPF2 in cells infected with influenza virus resulted in decreased expression of viral proteins and RNA. Furthermore, production of progeny virus was reduced by two logs in the multiple-cycle growth kinetics assay. We also found that DPF2 was involved in the replication of seasonal influenza A and B viruses. Because DPF2 plays a crucial role in the noncanonical NF-B pathway, which negatively regulates type I interferon (IFN) induction, we examined the relationship between DPF2 and IFN responses during viral infection. The results showed that knockdown of DPF2 resulted in increased expression of IFN-bbeta; and induced phosphorylation of STAT1 in infected cells. In addition, high levels of several cytokines/chemokines (interleukin-8 [IL-8], IP-10, and IL-6) and antiviral proteins (MxA and ISG56) were produced by DPF2 knockdown cells. In conclusion, we identified a novel host factor, DPF2, that is required for influenza virus to evade the host immune response and that may serve as a potential antiviral target.

    IMPORTANCE Influenza virus is responsible for seasonal epidemics and occasional pandemics and is an ongoing threat to public health worldwide. Influenza virus relies heavily on cellular factors to complete its life cycle. Here we identified a novel host factor, DPF2, which is involved in influenza virus infection. Our results showed that DPF2 plays a crucial role in the replication and propagation of influenza virus. DPF2 functions in the noncanonical NF-B pathway, which negatively regulates type I IFN induction. Thus, we investigated the relationship between the IFN response and DPF2 in influenza virus infection. Upon influenza virus infection, DPF2 dysregulated IFN-bbeta; induction and expression of cytokines/chemokines and antiviral proteins. This study provides evidence that influenza virus utilizes DPF2 to escape host innate immunity.

  • Tetherin Inhibits Cell-Free Virus Dissemination and Retards Murine Leukemia Virus Pathogenesis [Pathogenesis and Immunity]

  • The relative contributions of cell-free virion circulation and direct cell-to-cell transmission to retroviral dissemination and pathogenesis are unknown. Tetherin/Bst2 is an antiviral protein that blocks enveloped virion release into the extracellular milieu but may not inhibit cell-to-cell virus transmission. We developed live-cell imaging assays which show that tetherin does not affect Moloney murine leukemia virus (MoMLV) spread, and only minimally affects vesicular stomatitis virus (VSV) spread, to adjacent cells in a monolayer. Conversely, cell-free MLV and VSV virion yields and VSV spread to distal cells were dramatically reduced by tetherin. To elucidate the roles of tetherin and cell-free virions during in vivo viral dissemination and pathogenesis, we developed mice carrying an inducible human tetherin (hTetherin) transgene. While ubiquitous hTetherin expression was detrimental to the growth and survival of mice, restriction of hTetherin expression to hematopoietic cells gave apparently healthy mice. The expression of hTetherin in hematopoietic cells had little or no effect on the number of MoMLV-infected splenocytes and thymocytes. However, hTetherin expression significantly reduced cell-free plasma viremia and also delayed MoMLV-induced disease. Overall, these results suggest that MoMLV spread within hematopoietic tissues and cell monolayers involves cell-to-cell transmission that is resistant to tetherin but that virion dissemination via plasma is inhibited by tetherin and is required for full MoMLV pathogenesis.

    IMPORTANCE Retroviruses are thought to spread primarily via direct cell-to-cell transmission, yet many have evolved to counteract an antiviral protein called tetherin, which may selectively inhibit cell-free virus release. We generated a mouse model with an inducible tetherin transgene in order to study how tetherin affects retroviral dissemination and on which cell types its expression is required to do so. We first developed a novel in vitro live-cell imaging assay to demonstrate that while tetherin does indeed dramatically reduce cell-free virus spreading, it has little to no effect on direct cell-to-cell transmission of either vesicular stomatitis virus (VSV) or the retrovirus MoMLV. Using our transgenic mouse model, we found that tetherin expression on hematopoietic cells resulted in the specific reduction of MoMLV cell-free plasma viremia but not the number of infected hematopoietic cells. The delay in disease associated with this scenario suggests a role for cell-free virus in retroviral disease progression.

  • Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation [Structure and Assembly]

  • Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans. These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.

    IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.

  • Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice [Vaccines and Antiviral Agents]

  • Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2nndash;EHV-1nndash;gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2nndash;EHV-1nndash;gD vaccine and the commercially available vaccine Vetera EHVXP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals (P llt; 0.01 or P llt; 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2nndash;EHV-1nndash;gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P llt; 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P llt; 0.01 or P llt; 0.05). Vaccination with VC2nndash;EHV-1nndash;gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4+ T cells and CD8+ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.

    IMPORTANCE A novel virus-vectored VC2nndash;EHV-1nndash;gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.

  • Defining a Novel Role for the Coxsackievirus and Adenovirus Receptor in Human Adenovirus Serotype 5 Transduction In Vitro in the Presence of Mouse Serum [Virus-Cell Interactions]

  • Human adenoviral serotype 5 (HAdV-5) vectors have predominantly hepatic tropism when delivered intravascularly, resulting in immune activation and toxicity. Coagulation factor X (FX) binding to HAdV-5 mediates liver transduction and provides protection from virion neutralization in mice. FX is dispensable for liver transduction in mice lacking IgM antibodies or complement, suggesting that alternative transduction pathways exist. To identify novel factor(s) mediating HAdV-5 FX-independent entry, we investigated HAdV-5 transduction in vitro in the presence of serum from immunocompetent C57BL/6 or immunocompromised mice lacking IgM antibodies (Rag 2nndash;/nndash; and NOD-scid-gamma [NSG]). Sera from all three mouse strains enhanced HAdV-5 transduction of A549 cells. While inhibition of HAdV-5nndash;FX interaction with FX-binding protein (X-bp) inhibited transduction in the presence of C57BL/6 serum, it had negligible effect on the enhanced transduction observed in the presence of Rag 2nndash;/nndash; or NSG serum. Rag 2nndash;/nndash; serum also enhanced transduction of the FX binding-deficient HAdV-5HVR5*HVR7*E451Q (AdT*). Interestingly, Rag 2nndash;/nndash; serum enhanced HAdV-5 transduction in a FX-independent manner in CHO-CAR and SKOV3-CAR cells (CHO or SKOV3 cells transfected to stably express human coxsackievirus and adenovirus receptor [CAR]). Additionally, blockade of CAR with soluble HAdV-5 fiber knob inhibited mouse serum-enhanced transduction in A549 cells, suggesting a potential role for CAR. Transduction of HAdV-5 KO1 and HAdV-5/F35 (CAR binding deficient) in the presence of Rag 2nndash;/nndash; serum was equivalent to that of HAdV-5, indicating that direct interaction between HAdV-5 and CAR is not required. These data suggest that FX may protect HAdV-5 from neutralization but has minimal contribution to HAdV-5 transduction in the presence of immunocompromised mouse serum. Alternatively, transduction occurs via an unidentified mouse serum protein capable of bridging HAdV-5 to CAR.

    IMPORTANCE The intravascular administration of HAdV-5 vectors can result in acute liver toxicity, transaminitis, thrombocytopenia, and injury to the vascular endothelium, illustrating challenges yet to overcome for HAdV-5-mediated systemic gene therapy. The finding that CAR and potentially an unidentified factor present in mouse serum might be important mediators of HAdV-5 transduction highlights that a better understanding of the complex biology defining the interplay between adenovirus immune recognition and cellular uptake mechanisms is still required. These findings are important to inform future optimization and development of HAdV-5-based adenoviral vectors for gene therapy.

  • Group A Rotaviruses in Chinese Bats: Genetic Composition, Serology, and Evidence for Bat-to-Human Transmission and Reassortment [Genetic Diversity and Evolution]

  • Bats are natural reservoirs for many pathogenic viruses, and increasing evidence supports the notion that bats can also harbor group A rotaviruses (RVAs), important causative agents of diarrhea in children and young animals. Currently, 8 RVA strains possessing completely novel genotype constellations or genotypes possibly originating from other mammals have been identified from African and Chinese bats. However, all the data were mainly based on detection of RVA RNA, present only during acute infections, which does not permit assessment of the true exposure of a bat population to RVA. To systematically investigate the genetic diversity of RVAs, 547 bat anal swabs or gut samples along with 448 bat sera were collected from five South Chinese provinces. Specific reverse transcription-PCR (RT-PCR) screening found four RVA strains. Strain GLRL1 possessed a completely novel genotype constellation, whereas the other three possessed a constellation consistent with the MSLH14-like genotype, a newly characterized group of viruses widely prevalent in Chinese insectivorous bats. Among the latter, strain LZHP2 provided strong evidence of cross-species transmission of RVAs from bats to humans, whereas strains YSSK5 and BSTM70 were likely reassortants between typical MSLH14-like RVAs and human RVAs. RVA-specific antibodies were detected in 10.7% (48/448) of bat sera by an indirect immunofluorescence assay (IIFA). Bats in Guangxi and Yunnan had a higher RVA-specific antibody prevalence than those from Fujian and Zhejiang provinces. These observations provide evidence for cross-species transmission of MSLH14-like bat RVAs to humans, highlighting the impact of bats as reservoirs of RVAs on public health.

    IMPORTANCE Bat viruses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), Ebola, Hendra, and Nipah viruses, are important pathogens causing outbreaks of severe emerging infectious diseases. However, little is known about bat viruses capable of causing gastroenteritis in humans, even though 8 group A viruses (RVAs) have been identified from bats so far. In this study, another 4 RVA strains were identified, with one providing strong evidence for zoonotic transmission from bats to humans. Serological investigation has also indicated that RVA infection in bats is far more prevalent than expected based on the detection of viral RNA.

  • DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription [Genome Replication and Regulation of Viral Gene Expression]

  • African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (nndash;78.3%), and reduced transcription of a late viral gene (nndash;47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.

    IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

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    JVI Accepts: Articles Published Ahead of Print

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

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

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

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

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

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

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

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

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

  • A map of the arenavirus nucleoprotein-host protein interactome reveals that Junin virus selectively impairs the antiviral activity of PKR [PublishAheadOfPrint]

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

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

  • Reducing V3 antigenicity and immunogenicity on soluble, native-like HIV-1 Env SOSIP trimers [PublishAheadOfPrint]

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

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

  • Anti-HIV-1 ADCC antibodies following latency reversal and treatment interruption [PublishAheadOfPrint]

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

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

  • Uncovering the repertoire of endogenous flaviviral elements in Aedes mosquito genomes [PublishAheadOfPrint]

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

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

  • Heparan sulfate proteoglycan is an important attachment factor for cell entry of Akabane and Schmallenberg viruses [PublishAheadOfPrint]

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

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

  • Infectious Prions in the Pregnancy Microenvironment of CWD-infected Reeves' Muntjac Deer [PublishAheadOfPrint]

  • Ample evidence exists for the presence of infectious agents at the maternal-fetal interface, often with grave outcomes to the developing fetus (i.e. zika virus, brucella, cytomegalovirus, toxoplasma). While less studied, pregnancy-related transmissible spongiform encephalopathies (TSEs) have been implicated in several species, including humans. Our previous work has shown that prions can be transferred from mother-to-offspring resulting in the development of clinical TSE disease in offspring born to CWD-infected muntjac dams (64). We further demonstrated protein misfolding cyclic amplification (PMCA)-competent prions within the female reproductive tract and in fetal tissues harvested from CWD experimental and naturally-exposed cervids (64, 81). To assess whether the PMCA-competent prions residing at the maternal-fetal interface were infectious, and to determine if the real time quaking-induced conversion (RT-QuIC) methodology may enhance our ability to detect amyloid fibrils within the pregnancy microenvironment, we employed mouse bioassay and RT-QuIC. In this study we have demonstrated RT-QuIC seeding activity in uterus, placentome, ovary and amniotic fluid, but not allantoic fluids harvested from clinical CWD-infected Reeves' muntjac dams and some placentomes from pre-clinical CWD-infected dams. Prion infectivity was confirmed within the uterus, amniotic fluid and the placentome, the semipermeable interface that sustains the developing fetus, of CWD-infected dams. This is the first report of prion infectivity within the cervid pregnancy microenvironment, revealing a source of fetal CWD exposure prior to the birthing process, maternal grooming, or encounter with contaminated environments.

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

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

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

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

  • Ebola Virus Delta Peptide is a Viroporin [PublishAheadOfPrint]

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

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

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

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

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

  • Spontaneous mutation at amino acid 544 of the Ebola glycoprotein potentiates virus entry and selection in tissue culture [PublishAheadOfPrint]

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

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

  • Chikungunya virus overcomes polyamine depletion by mutation of nsP1 and the opal stop codon to confer enhanced replication and fitness [PublishAheadOfPrint]

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

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

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

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

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

  • Contribution of human lung parenchyma and leukocyte influx to oxidative stress and immune-mediated pathology following Nipah infection. [PublishAheadOfPrint]

  • Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause a fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD scid gamma mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the Bone marrow, Liver, and Thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections.

    Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system reconstituted mice in order to identify the overall effect of immune cells in NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects, irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in the lung grafts. In addition, the presence of human leukocytes increased expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI, and informs efforts to identify therapeutic targets.

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

  • Improved prefusion stability, optimized codon-usage, and augmented virion packaging enhance the immunogenicity of respiratory syncytial virus (RSV) fusion protein in a vectored vaccine candidate [PublishAheadOfPrint]

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

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

  • Discovery of a Prefusion RSV F-Specific Monoclonal Antibody that Provides Greater in vivo Protection than the Murine Precursor of Palivizumab [PublishAheadOfPrint]

  • Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the RSV F glycoprotein, is the only prophylactic agent approved for RSV. However, its relatively low neutralizing potency and high cost has limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high potency neutralizing antibody, 5C4, that specifically recognizes antigenic site OOslash; at the apex of the pre-F trimer. We comparednndash;in vitro and in vivonndash;the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 have similar pre-F binding affinities, 5C4 neutralizing activity is nearly 50-fold greater than that of 1129 in vitro. In BALB/c mice, 5C4 reduced peak RSV titers 1000-fold more than 1129 in both upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site OOslash; are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site OOslash;-specific antibodies may be useful for RSV prevention or therapy and support the use of pre-F as a vaccine antigen.

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

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

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

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

  • Spontaneous and vaccine-induced clearance of Mus musculus Papillomavirus type 1 (MmuPV1/MusPV1) infection [PublishAheadOfPrint]

  • Mus musculus Papillomavirus1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not common laboratory strains. To facilitate study of immune control, we sought an outbred and immune competent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) by scarification on their tail with MmuPV1 resulted in three clinical outcomes: 1) persistent (ggt;2 months) papillomas (~20%), 2) transient papillomas that spontaneously regress typically within 2 months (~15%), 3) no visible papillomas and viral clearance (~65%). SKH1 mice with persistent papillomas were treated using a candidate preventive/therapeutic naked DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and E7 (mE7) early proteins and residues 11-200 of late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and failure of disease to recur after CD3 T cell depletion. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice, although significantly lower in mice that initially presented with persistent warts as compared with those that spontaneously cleared their infection. An HPV16-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with HPV16 pseudovirus. Thus MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearance of HPV-related disease.

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

  • Transposon mutagenesis of the Zika virus genome highlights regions essential for RNA replication and restricted for immune evasion [PublishAheadOfPrint]

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

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

  • Central Nervous System Infection with Borna Disease Virus Causes Kynurenine Pathway Dysregulation and Neurotoxic Quinolinic Acid Production [PublishAheadOfPrint]

  • Central nervous system infection of neonatal and adult rats with Borna disease virus (BDV) results in neuronal destruction and behavioral abnormalities with differential immune-mediated involvement. Neuroactive metabolites generated from the kynurenine pathway of tryptophan degradation have been implicated in several human neurodegenerative disorders. Here we report that brain expression of key enzymes in the kynurenine pathway are significantly, but differentially, altered in neonatal and adult rats with BDV infection. Gene expression analysis of rat brains following neonatal infection showed increased expression of kynurenine amino transferase II (KATII) and kynurenine-3-monooxygenase (KMO) enzymes. Additionally, indoleamine 2,3-dioxygenase (IDO) expression was only modestly increased in a brain region- and temporally-dependent manner in neonatally infected rats; however, its expression was highly increased in adult infected rats. The most dramatic impact on gene expression was seen for KMO, whose activity promotes the production of neurotoxic quinolinic acid. KMO expression was persistently elevated in brain regions of both neonatally and adult infected rats, with increases reaching up to 86-fold. KMO protein levels were increased in neonatally infected rats and colocalized with neurons, the primary target cells of BDV infection. Furthermore, quinolinic acid was elevated in neonatally infected rat brains. We further demonstrate increased expression of KATII and KMO, but not IDO, in vitro in BDV infected C6 astroglioma cells. Our results suggest that BDV directly impacts the kynurenine pathway, an effect that may be exacerbated by inflammatory responses in immunocompetent hosts. Thus, experimental models of BDV infection may provide new tools for discriminating virus-mediated vs. immune-mediated impacts on the kynurenine pathway and their relative contribution to neurodegeneration.

    IMPORTANCE BDV causes persistent, noncytopathic infection in vitro, yet still elicits widespread neurodegeneration of infected neurons in both immunoincompetent and immunocompetent hosts. Here we show that BDV infection induces expression of key enzymes of the kynurenine pathway in brains of neonatally and adult infected rats and cultured astroglioma cells, shunting tryptophan degradation toward the production of neurotoxic quinolinic acid. Thus, our findings newly implicate this metabolic pathway in BDV-induced neurodegeneration. Given the importance of the kynurenine pathway in a wide range of human infections and neurodegenerative and neuropsychiatric disorders, animal models of BDV infection may serve as important tools for contrasting direct viral and indirect anti-viral immune-mediated impacts on kynurenine pathway dysregulation and ensuing neurodevelopmental and neuropathological consequences.

  • A novel A(H7N2) influenza virus isolated from a veterinarian caring for cats in a New York City animal shelter causes mild disease and transmits poorly in the ferret model. [PublishAheadOfPrint]

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

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

  • Crimean-Congo hemorrhagic fever virus nucleocapsid protein augments mRNA translation [PublishAheadOfPrint]

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

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

  • Deletion of the Vaccinia B1 Kinase Reveals Essential Functions of this Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2 [PublishAheadOfPrint]

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

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

  • The influenza A virus NS1 protein promotes efficient nuclear export of unspliced viral M1 mRNA [PublishAheadOfPrint]

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

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

  • A point mutation in the Rhesus rotavirus VP4 protein generated through rotavirus reverse genetics system attenuates the murine model of biliary atresia [PublishAheadOfPrint]

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

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

  • Hepatitis C virus lipoviroparticles (HCV-LVP) assemble in the endoplasmic reticulum (ER) and bud off from the ER to Golgi in COPII vesicles [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity leading to the notion that HCV co-opts the secretion of very-low density lipoprotein (VLDL) for its egress. However, our understanding of the mechanisms involved in virus particle assembly and egress, are still elusive. Biogenesis of VLDL particle occurs in the ER followed by subsequent lipidation in the ER and Golgi. Secretion of mature VLDL particles occurs through the Golgi secretory pathway. The HCV virions are believed to latch on or fuse with the nascent VLDL particle in either ER or Golgi compartment resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion we studied the HCV particles budding from the ER en route to the Golgi in the COP-II vesicles. Biophysical characterization of COP-II vesicles fractionated on iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and co-fractionates with apolipoproteins (apo) B, apoE, HCV core and envelope proteins. Electron microscopy of immuno-gold labeled micro-sections revealed that HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec-31 coated COP-II vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV-LVPs assemble in the ER and are transported to the Golgi in the COPII vesicles to embark on the Golgi secretory route.

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

  • Differential disease susceptibility in experimentally reptarenavirus infected boa constrictors and ball pythons [PublishAheadOfPrint]

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

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

  • Natural variation of Epstein-Barr virus genes, proteins and pri-miRNA (revised) [PublishAheadOfPrint]

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

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

  • Virus-like vesicles of Kaposi's sarcoma-associated herpesvirus activate lytic replication through triggering differentiation signaling [PublishAheadOfPrint]

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

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

  • Cell cycle-dependent expression of AAV2 Rep in HSV-1 co-infections gives rise to a mosaic of cells replicating either AAV2 or HSV-1 [PublishAheadOfPrint]

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

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

  • Molluscum Contagiosum Virus MC159 abrogates cIAP1--NEMO interactions and inhibits NEMO polyubiquitination [PublishAheadOfPrint]

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

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

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

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

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

  • Attenuation of Foot-and-Mouth Disease Virus by Engineering Viral Polymerase Fidelity [PublishAheadOfPrint]

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

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

  • ROS-mediated JNK activation contributes to HBx-induced autophagy via regulating Beclin-1/Bcl-2 interaction [PublishAheadOfPrint]

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

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

  • Structural Definition of Duck MHC class I Molecules that might explain efficient CTL Immunity against Influenza A Virus [PublishAheadOfPrint]

  • A single dominantly expressed allele of the major histocompatibility complex class I (MHC I) may be responsible for the duck's high tolerance to the highly pathogenic (HP) influenza A virus (IAV) compared to the chicken's lower tolerance. In this study, the crystal structures of duck MHC I (Anpl-UAA*01) and duck bbeta;2m with two peptides from the H5N1 strains were determined, respectively. Two remarkable features were found to distinguish the Anpl-UAA*01 complex from other known MHC I structures. A disulfide bond formed by Cys95 and Cys112 and connecting the bbeta;5 and bbeta;6 sheets at the bottom of peptide binding groove (PBG) in Anpl-UAA*01 complex, which can enhance IAV peptides binding, was identified. Moreover, the interface area between duck MHC I and bbeta;2m was found to be larger than in other species. In addition, the two IAV peptides that display distinctive conformations in the PBG, B, and F pockets act as the primary anchor sites. 31 IAV peptides were used to verify the peptide binding motif of Anpl-UAA*01, and the results confirmed that the peptide binding motif is similar to HLA-A*0201. Basing on this motif, approximate 600 peptides from the IAV strains were partially verified as the candidate epitope-peptides for Anpl-UAA*01, which is a far greater number than that for chicken BF2*2101 and BF2*0401 molecules. Extensive IAV peptide binding should allow for ducks with this Anpl-UAA*01 haplotype to resist IAV infection.

    IMPORTANCE Ducks are natural reservoirs of the influenza A virus (IAV) and are more resistant to the IAV than chickens. Both ducks and chickens express only one dominant MHC I locus providing resistance to the virus. To investigate how MHC I provides IAV resistance, crystal structures of the dominantly expressed duck MHC class I (pAnpl-UAA*01) with two IAV peptides were determined. A disulfide bond was identified in the peptide binding groove that can facilitate Anpl-UAA*01 binding to IAV peptides. Anpl-UAA*01 has a much wider recognition spectrum of IAV epitope-peptides compared to chickens. The IAV peptides bound by Anpl-UAA*01 display distinctive conformations that can help induce an extensive cytotoxic T lymphocyte (CTL) response. In addition, the interface area between the duck MHC I and bbeta;2m is larger than in other species. These results indicate that HP-IAV resistance in ducks is due to extensive CTL responses induced by MHC I.

  • Congenital HCMV infection and the enigma of maternal immunity [PublishAheadOfPrint]

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

  • Dok-1 and Dok-2 are required to maintain herpes simplex virus 1-specific CD8+ T cells in a murine model of ocular infection [PublishAheadOfPrint]

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

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

  • Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in Both Host and Viral Genomes [PublishAheadOfPrint]

  • Rta, an Epsteinnndash;Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1 and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1 and JUN are common in: (1) presence of CpG islands, (2) strong ChIP signals of CCCTC-binding factor (CTCF), (3) at least one Rta binding site is found. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1 and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Thereby, via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.

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

  • The C-terminus of the herpes simplex virus pUL25 protein is required for release of viral genomes from capsids bound to nuclear pores. [PublishAheadOfPrint]

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

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

  • Heparanase upregulation contributes to porcine reproductive and respiratory syndrome virus release [PublishAheadOfPrint]

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

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

  • Roles of M1 and M2 macrophages in herpes simplex virus-1 infectivity [PublishAheadOfPrint]

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

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

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

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

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

  • Development of clade-specific and broadly reactive live attenuated influenza virus vaccines against rapidly evolving H5 subtype viruses. [PublishAheadOfPrint]

  • We have developed pandemic live attenuated influenza vaccines (pLAIV) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted (ca) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (2.2.1.1) of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt that prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs: first to modify the hemagglutinin gene of a highly pathogenic wild-type (wt) clade 2.2.1.1 virus A/Egypt/N03434/2009 (Egy/09) (H5N1) with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca. The second approach was to select a low pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses and contained a novel NA subtype for humans. We selected the low pathogenicity A/duck/Hokkaido/69/2000 (H5N3) virus (dk/Hok/00) for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific to the gs/GD lineage viruses but did not neutralize recent North American isolates (clade 2.3.4.4), whereas antisera from dk/Hok/69 ca vaccinated ferrets cross-reacted with clade 2.3.4.4 and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses.

    IMPORTANCE: In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed using two different approaches: one targeted a specific circulating strain in Egypt and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches and the data support further development of both vaccine viruses to optimally prepare for further spread of clade 2.2.1 or 2.3.4.4 viruses.

  • Molluscum Contagiosum Virus protein MC005 inhibits NF{kappa}B activation by targeting NEMO-regulated IKK activation [PublishAheadOfPrint]

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

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

  • Deletion of the K1L gene results in a vaccinia virus that is less pathogenic due to muted innate immune responses, yet still elicits protective immunity [PublishAheadOfPrint]

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

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

  • A naturally occurring recombinant enterovirus expresses a torovirus deubiquitinase [PublishAheadOfPrint]

  • Enteroviruses are implicated in a wide range of diseases in human and animals. In this study, a novel enterovirus (species G; EVG 08/NC_USA/2015) was isolated from a diagnostic sample of neonatal pig diarrhea case and identified using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582 nucleotide insertion, flanked by 3Cpro cleavage sites at 5rrsquo; - and 3rrsquo; - ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54-68% amino acid identity to torovirus (ToV) papain-like protease (PLP). Structural homology modeling predicts that this protease adopts a fold and catalytic site characteristic of minimal PLP catalytic domains. The structure is similar to those of core catalytic domains of the foot-and-mouth disease leader protease and coronavirus PLPs, which act as de-ubiquitinating and deISGylating enzymes on host cell substrates. Importantly, recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities, and demonstrated the capability to suppress IFN-bbeta; expression. Using reverse genetics, we generated ToV-PLP knockout recombinant virus. Compared to wild type virus, ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.

    IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered as a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order picornavirales) and Torovirus (order Nidovirales). This naturally occurring recombinant event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights on how gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.

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

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

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

  • Evolution and antiviral specificity of interferon-induced Mx proteins of bats against Ebola-, Influenza-, and other RNA viruses. [PublishAheadOfPrint]

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

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

  • Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets [PublishAheadOfPrint]

  • We previously isolated a porcine epidemic diarrhea virus (PEDV) strain PC177, by blind serial passaging the intestinal contents of a diarrheic piglet in Vero cell culture. Compared with a US highly virulent PEDV strain PC21A, the tissue culture-adapted PC177 (TC-PC177) contains a 197 aa-deletion in the N-terminal domain of the spike protein. We orally inoculated neonatal, conventional suckling piglets with TC-PC177 or PC21A to compare the pathogenicity. Within 7 days post-inoculation, TC-PC177 caused mild diarrhea and lower fecal viral RNA shedding, with no mortality, whereas PC21A caused severe clinical signs and 55% mortality. To investigate whether infection of TC-PC177 can induce cross-protection against highly virulent PEDV strain challenge, all the surviving piglets were challenged with PC21A at 3 weeks post-inoculation. Compared with 100% protection in piglets initially inoculated with PC21A, 88% and 100% TC-PC177- and mock-inoculated piglets had diarrhea following challenge, respectively, indicating incomplete cross-protection. To investigate whether this 197 aa-deletion was the determinant for the attenuation of TC-PC177, we generated a mutant (icPC22A-S1197) bearing the 197 aa-deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (icPC22A). In neonatal gnotobiotic pigs, the icPC22A-S1197 virus caused mild to moderate diarrhea, lower titers of viral shedding and no mortality, whereas the icPC22A virus caused severe diarrhea and 100% mortality. Our data indicate that deletion of this 197 aa-fragment in the spike protein can attenuate a highly virulent PEDV, but the virus may lose important epitopes for inducing robust protective immunity.

    IMPORTANCE The emerging, highly virulent PEDV strains have caused substantial economic losses worldwide. However, the virulence determinants are not established. In this study, we found a 197 aa-deletion in the N-terminal region of the S protein did not alter virus (TC-PC177) tissue tropism, but reduced the virulence of a highly virulent PEDV strain PC22A in neonatal piglets. We also demonstrated that the primary infection of TC-PC177 failed to induce complete cross-protection against challenge by the highly virulent PEDV PC21A, suggesting that the 197 aa-region may contain important epitopes for inducing protective immunity. Our results provide an insight into the role of this large deletion in virus propagation and pathogenicity. In addition, the reverse genetics platform of PC22A strain was further optimized for the rescue of recombinant PEDV viruses in vitro. This breakthrough allows us to investigate other virulence determinants of PEDV strains and will provide knowledge to better control PEDV infections.

  • Induction of a Tier-1-like Phenotype in Diverse Tier-2 Isolates by Agents that Guide HIV-1 Env to Perturbation-sensitive, Non-native States [PublishAheadOfPrint]

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

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

  • Sensing of HIV-1 entry triggers a type I interferon response in human primary macrophages [PublishAheadOfPrint]

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

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

  • Human sera collected during 1979-2010 possess blocking antibody titers to pandemic GII.4 noroviruses isolated during 3 decades [PublishAheadOfPrint]

  • The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII.4 noroviruses isolated in last 4 decades.

    In total 268 sera were investigated for blocking titers (BT50 values) of virus-like particles (VLPs) to pig gastric mucin (PGM) using 4 VLPs that represent different GII.4 norovirus variants identified between 1987-2012.

    Pre- and post-pandemic sera (sera collected before- and after- isolation of reference NoV strain) efficiently prevented binding of VLP strains MD145 (1987), Grimsby (1996) and Houston (2002) but not the Sydney (2012) strain to PGM. No statistically significant difference in virus blocking titer was observed between pre- and post-pandemic sera. Moreover, paired sera showed that blocking titers of gge;160 were maintained over a 6-year period against MD145, Grimsby and Houston VLPs. Significantly higher serum blocking titers (GMT 1704) were found among IgA-deficient individuals in comparison to healthy blood donors (GMT 90.9) (pllt;0.0001). The observation that pre-pandemic sera possess robust virus blocking capacity to viruses identified decades later suggests a common attachment factor at least until year 2002. Our results indicate that serum IgG possesses antibody-blocking capacity and that blocking titers can be maintained for at least 6 years against 3 decades of pandemic GII.4 NoV.

    IMPORTANCE Human noroviruses (NoVs) are the major cause of acute gastroenteritis worldwide. Histo blood group antigens (HBGA), in saliva and gut recognize NoV and are the proposed ligands to facilitate infection. Polymorphisms in HBGA genes and particular a nonsense mutation in FUT2 (G428A), result in resistance to global dominating GII.4 NoV. Emergence of new pandemic GII.4 strains occurs with several years intervals and is proposed to be attributed to epochal evolution including amino acid changes and immune evasion. However, it remains unclear whether exposure to a previous pandemic strain stimulates immunity to a pandemic strain identified decades later. We found that pre-pandemic sera possess robust virus blocking capacity to viruses identified several decades later. We also show that sera lacking IgA antibodies is sufficient to block NoV VLP binding to HBGAs. This is essential, considering that 1 in every 600 Caucasian children is IgA deficient.

  • Preservation of peripheral T follicular helper cell function in HIV controllers [PublishAheadOfPrint]

  • The maturation process of high-affinity antibodies is a result of intricate interactions between B cells and follicular helper T (Tfh) cells occurring in lymphoid germinal centers. HIV infection induces significant chronic immune activation, phenotypic skewing, and inflammation driven by years of continuous viral replication. High levels of viremia as well as immune activation and dysfunction have been demonstrated to have a perturbing impact on the B cell memory compartment and contribute to B cell exhaustion. Counterintuitively, the same factors associated with perturbation of the B cell compartment seem to be favorable factors for the generation of highly affinity matured Env-specific antibodies in a minority of HIV-infected individuals. Thus, understanding the impact of HIV antigenemia on B cells and Tfh cell interactions warrant further exploration. We therefore studied immunophenotypes of HIV-specific B cells in individuals with differing levels of viral control using HIV Env gp120-probes and characterized the functionality of matched T cells in peripheral blood. While CXCR5+CD4+ T cells were significantly diminished in HIV progressors, we found that a small subset of gp120-specific IL-21-secreting CXCR5+CD4 T cells were significantly associated with gp120-specific B cell frequencies. In contrast, neither bulk CXCR5+CD4 T cells nor other HIV antigen specificities were associated with gp120-specific B cell levels. HIV-specific B cells derived from elite controllers displayed greater amounts of gp120-specific B cells in the resting-memory subset whereas HIV-specific B cells in progressors accumulated in tissue-like and activated memory subsets. Furthermore, elite controller-derived CXCR5+CD4 T cells showed a stronger ex vivo capacity to induce B cell maturation and immunoglobulin class switching compared to HIV progressors.

    IMPORTANCE Dissecting the factors that are involved in B cell maturation and antibody development is important to understand for HIV vaccine design. Here we found that HIV Env-specific CXCR5+ CD4+T cells that are secreting Interleukin-21 are strongly associated with B cell memory phenotypes and function. Moreover, we found that the immune responses of HIV controllers showed intrinsically better helper activity than HIV progressors.

  • Cell culture systems to study Human Herpesvirus 6A/B Chromosomal Integration [PublishAheadOfPrint]

  • Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5h) cells were processed to single-cell cloning and analyzed for chromosomally-integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using qPCR, droplet digital PCR and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase positive (HeLa, MCF-7, HCT-116, HEK293T) and telomerase negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of ciHHV-6A+/B+ cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes including U39, U90 and U100 without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression and release of infectious HHV-6A/B from the integrated state.

    IMPORTANCE The analysis and understanding of human herpesviruses 6A and 6B (HHV-6A/B) genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell-culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.

  • SUPPRESSOR ANALYSIS OF THE FUSOGENIC LAMBDA SPANINS [PublishAheadOfPrint]

  • The final step of lysis in infections of E. coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and has been proposed to cause fusion of the inner and outer membrane. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque-formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele-specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo. Unlike the wild type, in which lysis occurs while the cells retain rod-shape, revertant alleles with second site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled-coil restores function for defective spanin alleles by increasing the conformational freedom of the complex, at the cost of its normal all-or-nothing functionality.

    IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membrane during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class-I viral fusion proteins. While the pre- and post-fusion structures of model fusion systems have been well characterized, intermediate structure(s) formed during the fusion reaction remain elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable only few such investigations have been reported. Here we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class-I-like complex and also the first such analysis of a prokaryote membrane fusion system.

  • Stabilization of telomere G-quadruplexes interferes with human herpesvirus 6A chromosomal integration [PublishAheadOfPrint]

  • Human herpesvirus 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6 integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevent telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. Next, we investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A ability to integrate its genome into host chromosomes. Incubation of telomerase expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (pllt;0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affect the generation of cellular clones containing integrated HHV-6A.

    Importance Human herpesvirus 6A/B (HHV-6A/B) can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of varying length (up to several kilobases) and end with a single-stranded 3rrsquo; extension. To avoid recognition and induce a DNA-damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure referred to as G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6. This study is the first of its kind and suggests that telomerase activity is likely essential to restore a functional telomere of adequate length following HHV-6A integration.

  • Serum Metabolomics Investigation of Humanized Mouse Model with Dengue Infection [PublishAheadOfPrint]

  • Dengue is an acute febrile illness caused by dengue virus (DENV) and a major cause of morbidity and mortality in tropical and subtropical regions of the world. The lack of an appropriate small-animal model of dengue infection has greatly hindered the study of dengue pathogenesis and the development of therapeutics. In this study, we conducted a mass spectrometry-based serum metabolic profiling from a humanized mice (humice) model with DENV serotype 2 infection at 0, 3, 7, 14 and 28 days post infection (dpi). Forty-eight differential metabolites were identified, including fatty acids, purines and pyrimidines, acylcarnitines, acylglycines, phospholipids, sphingolipids, amino acid and derivatives, free fatty acids, bile acid, etc. These metabolites showed a reversible change trend - most were significantly perturbed at 3 or 7 dpi and returned to control levels at 14 or 28 dpi, indicating that these metabolites might serve as prognostic markers of the disease in humice. Major perturbed metabolic pathways included purine and pyrimidine metabolism, fatty acid bbeta;-oxidation, phospholipid catabolism, arachidonic acid and linoleic acid metabolism, sphingolipid metabolism, tryptophan metabolism, phenylalanine metabolism, lysine biosynthesis and degradation, bile acid biosynthesis, etc. Most of these disturbed pathways are similar to our previous metabolomics findings in a longitudinal cohort of adult human dengue patients across different infection stages. Our analyses revealed the commonalities of host responses between humice and human to DENV infections and suggested that humice could be a useful small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics.

    Importance: Dengue virus is the most widespread arbovirus, causing an estimated 390 million dengue infections worldwide every year. As yet, there is currently no effective treatment against the disease, and the lack of an appropriate small-animal model of dengue infection has greatly increased the challenges in the study of dengue pathogenesis and the development of therapeutics. Metabolomics provides global views of small molecule metabolites and is a useful tool of finding metabolic pathways related to disease processes. Here we conducted serum metabolomics study on a humanized mice model, which has significant levels of human platelets, monocytes/macrophages, and hepatocytes, with dengue infection. Forty-eight differential metabolites were identified, and the underlying perturbed metabolic pathways are quite similar to the pathways altered in dengue patients in previous metabolomics studies, indicating that humanized mice could be a highly relevant small-animal model for the study of dengue pathogenesis and the development of dengue therapeutics.

  • Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly [PublishAheadOfPrint]

  • Eastern equine encephalitis virus (EEEV) is a representative member of the New World alphaviruses. It is pathogenic for a variety of vertebrate hosts, in which EEEV induces highly debilitating disease with frequent lethal outcome. Despite a significant public health threat, the molecular mechanism of EEEV replication and interaction with hosts is poorly understood. Our previously published data and those of other teams have demonstrated that hypervariable domains (HVDs) of alphavirus nsP3 protein interact with virus-specific host factors and play critical roles in assembly of viral replication complexes (vRCs). The most abundantly represented HVD-binding proteins are the FXR and G3BP family members. FXR proteins drive the assembly of vRCs of Venezuelan equine encephalitis virus (VEEV), and G3BPs were shown to function in vRC assembly in replication of chikungunya and Sindbis viruses. Our new study demonstrates that EEEV exhibits a unique level of redundancy in the use of host factors in RNA replication. EEEV efficiently utilizes both the VEEV-specific FXR and the Old World alphavirus-specific G3BP protein families. Lack of interaction with either FXRs or G3BPs does not affect vRC formation; however, removal of EEEV's ability to interact with both protein families has deleterious effect on virus growth. Other identified EEEV nsP3 HVD-interacting host proteins are also capable of supporting EEEV replication, albeit with dramatically lower efficiency. The ability to use a wide range of host factors with redundant functions in vRC assembly and function provides a plausible explanation for efficient replication of EEEV and may contribute to highly pathogenic phenotype.

    IMPORTANCE Eastern equine encephalitis virus (EEEV) is one of the most pathogenic New World alphaviruses. Despite the continuous public health threat, to date, the molecular mechanisms of its very efficient replication and high virulence are not sufficiently understood. The results of this new study demonstrate that North American EEEV exhibits high level of redundancy in using host factors in replication complex assembly and virus replication. The hypervariable domain of EEEV nsP3 protein interacts with all of the members of FXR and G3BP protein families, and only lack of interaction with both protein families strongly affects virus replication rates. Other identified HVD-binding factors are also involved in EEEV replication, but their roles are not as critical as those of FXRs and G3BPs. The new data present a plausible explanation for exceptionally high replication rates of EEEV, and suggest a new means of its attenuation and targets for screening of antiviral drugs.

  • Subversion of host responses to energy insufficiency by Us3 supports HSV-1 replication during stress [PublishAheadOfPrint]

  • Cellular stress responses to energy insufficiency can impact virus reproduction. In particular, activation of the host AMP-activated protein kinase (AMPK) by low energy could limit protein synthesis by inhibiting mTORC1. Although many herpesviruses, including herpes simplex virus-1 (HSV-1), stimulate mTORC1, how HSV-1-infected cells respond to energy availability, a physiological indicator regulating mTORC1, has not been investigated. In addition, the impact of low energy stress on productive HSV-1 growth and viral genetic determinants potentially enabling replication under physiological stress remain undefined. Here, we demonstrate that mTORC1 activity in HSV-1-infected cells is largely insensitive to stress induced by simulated energy insufficiency. Furthermore, resistance of mTORC1 activity to low energy-induced stress, while not significantly influenced by the HSV-1 UL46-encoded PI3-kinase-Akt activator, was dependent upon the ser/thr kinase activity of Us3. A Us3-deficient virus was hypersensitive to low energy-induced stress, as infected cell protein synthesis and productive replication were reduced compared to cells infected with a Us3-expressing virus. Although Us3 did not detectably prevent energy stress-induced AMPK activation, it enforced mTORC1 activation despite the presence of activated AMPK. In the absence of applied low energy stress, AMPK activity in infected cells was restricted in a Us3-dependent manner. This establishes that the Us3 kinase not only activated mTORC1, but additionally enabled sustained mTORC1 signaling during simulated energy insufficiency that would otherwise restrict protein synthesis and virus replication. Moreover, it identifies the alpha-herpesvirus specific Us3 kinase as an mTORC1 activator that subverts the host cell energy-sensing program to support viral productive growth irrespective of physiological stress.

    IMPORTANCE Like all viruses, herpes simplex virus type-1 (HSV-1) reproduction relies upon numerous host energy intensive processes, the most demanding of which is protein synthesis. In response to low energy, the cellular AMP-activated protein kinase (AMPK) triggers a physiological stress response that antagonizes mTORC1, a multi-subunit host kinase that controls protein synthesis. This could restrict virus protein production and growth. Here, we establish that the HSV-1 Us3 protein kinase subverts the normal response to low energy-induced stress. While Us3 doesn't prevent AMPK activation by low energy, it enforces mTORC1 activation and overrides a physiological response that couples energy availability and protein synthesis. These results help explain how reproduction of HSV-1, a ubiquitous, medically significant human pathogen causing a spectrum of diseases ranging from the benign to life threatening, occurs during physiological stress. This is important because HSV-1 reproduction triggered by physiological stress is characteristic of reactivation of life-long latent infections.

  • CXCL10/CXCR3-Dependent Mobilization of HSV-Specific CD8+ TEM and CD8+ TRM Cells within Infected Tissues Allows Efficient Protection Against Recurrent Herpes Infection and Disease [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) establishes latency within the sensory neurons of the trigeminal ganglia (TG). HSV-specific memory CD8+ T cells play a critical role in preventing HSV-1 reactivation from TG and subsequent virus shedding in tears that trigger recurrent corneal herpetic disease. The CXCL10/CXCR3 chemokine pathway promotes T cell immunity to many viral pathogens, but its importance in CD8+ T cell immunity to recurrent herpes has been poorly elucidated. In this study, we determined how the CXCL10/CXCR3 pathway affects TG- and cornea-resident CD8+ T cell responses to recurrent ocular herpes infection and disease using a well-established murine model in which HSV-1 reactivation was induced from latently infected TG by UV-B light. Following UV-B induced HSV-1 reactivation; a significant increase in both the number and the function of HSV-specific CXCR3+CD8+ T cells was detected in TG and cornea of protected B6 mice, but not in TG and cornea of non-protected CXCL10-/- or CXCR3-/- deficient mice. This increase was associated with a significant reduction in both virus shedding and recurrent corneal herpetic disease. Furthermore, delivery of exogenous CXCL10 chemokine in TG of CXCL10-/- mice, using the neurotropic AAV8 vector, boosted the number and function of effector memory (TEM) and tissue-resident memory (TRM), but not of central memory (TCM) CD8+ T cells, locally within TG, and improved protection against recurrent herpes infection and disease in CXCL10-/- deficient mice. These findings demonstrate that the CXCL10/CXCR3 chemokine signaling pathway is critical in shaping CD8+ Tcell immunity, locally within latently infected tissues, which protects against recurrent herpes infection and disease.

    IMPORTANCE We determined how the CXCL10/CXCR3 pathway affects CD8+ T cell responses to recurrent ocular herpes infection and disease. Using a well-established murine model, in which HSV-1 reactivation was induced by UV-B from latently infected trigeminal ganglia, we demonstrated that lack of either CXCL10 chemokine or its CXCR3 receptor compromised the mobilization of functional CD8+ TEM and CD8+ TRM cells within latently infected trigeminal ganglia following virus reactivation. This lack of T cell mobilization was associated with an increase in recurrent ocular herpes infection and disease. Inversely, augmenting the amount of CXCL10 in trigeminal ganglia of latently infected CXCL10 deficient mice significantly restored the number of local antiviral CD8+ TEM and CD8+ TRM cells associated with protection against recurrent ocular herpes. Based on these findings, a novel "prime/pull" therapeutic ocular herpes vaccine strategy is proposed and discussed.

  • Rubicon modulates antiviral type I IFN signaling by targeting IRF3 dimerization [PublishAheadOfPrint]

  • Rubicon is a part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon TLR stimulation, and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon responses remains largely unknown. Here, we report that Rubicon acts as a negative regulator for virus-triggered IFN signaling. Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon has the opposite effect. Rubicon specifically interacts with the IRF association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response. Thus, our findings suggest that the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral responses, providing a novel cellular mechanism of IRF3 inhibition.

    IMPORTANCE The type I IFN system is a critical innate immune response that protects organisms against virus infection. However, type I IFN signaling must be tightly regulated to avoid excessive production of IFNs. Hence, negative regulatory mechanisms for type I IFN signaling are important, and to date, several related molecules have been identified. Here, we show that Rubicon is a major negative regulator of type I IFN signaling, and unlike previous reports of cellular molecules that inhibit IRF3 activation via proteasomal degradation or dephosphorylation of IRF3, we show that Rubicon interacts with IRF3, and ultimately this interaction leads to inhibit the dimerization of IRF3. Thus, we identified a novel negative regulator of type I IFN signaling pathways and a novel cellular mechanism of IRF3 inhibition. The results of this study will increase our understanding of the role of negative feedback mechanisms that regulate type I IFN signaling and maintain immune homeostasis.

  • Antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy versus Venezuelan equine encephalitis virus [PublishAheadOfPrint]

  • Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus that causes low mortality but high morbidity in humans. In addition to natural outbreaks, there is potential for exposure to VEEV via aerosolized virus particles. There are currently no FDA-licensed vaccines or antiviral therapies for VEEV. Passive immunotherapy is an approved method used to treat individuals against several pathogens and toxins. Human polyclonal antibodies (pAbs) are ideal, but this is dependent upon serum from convalescent human donors, which is in limited supply. Non-human derived pAbs can have serious immune-reactivity complications, and when "humanized" these antibodies may exhibit reduced neutralization efficiency. To address these issues, transchromosomic (Tc) bovines have been created that can produce potent neutralizing human antibodies in response to hyperimmunization. In the current studies, we have immunized these bovines with different VEEV immunogens and evaluated the protective efficacy of purified preparations of the resultant human polyclonal antisera against low and high dose VEEV challenge. The studies demonstrate that prophylactic or therapeutic administration of the polyclonal antibody preparations (TcpAbs) can protect mice against lethal subcutaneous or aerosol challenge with VEEV. Furthermore, significant protection can be conferred against unrelated co-infecting viral pathogens by combining individual virus-specific TcpAb preparations.

    IMPORTANCE With the globalization and spread or potential aerosol release of emerging infectious diseases, it will be critical to develop platforms able to produce therapeutics in a short time-frame. Using a transchromosomic (Tc) bovine platform, it is theoretically possible to produce antigen-specific highly neutralizing therapeutic polyclonal human antibody (TcpAb) preparations in six months or less. In this study, we demonstrate that Tc bovine-derived Venezuelan equine encephalitis virus (VEEV)-specific TcpAb are highly effective against VEEV infection that mimics not only the natural route of infection, but also infection via an aerosol exposure. Additionally, we show that combinatorial TcpAb preparations can be used to treat co-infections with divergent pathogens, demonstrating that the Tc bovine platform could be beneficial in areas where multiple infectious diseases contemporaneously or in the case of multipathogen release.

  • Immune escape variants of H9N2 influenza viruses containing deletions at the haemagglutinin receptor binding site retain fitness in vivo and display enhanced zoonotic characteristics [PublishAheadOfPrint]

  • H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa where they pose a threat to human health, both as zoonotic agents and as potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions, however vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza antigen, haemagglutinin (HA). Using selection with HA specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBS). Here, we analysed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that the mutant viruses with RBS deletions are able to escape polyclonal antisera binding and are able to infect and transmit between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells, however the mutant HAs also displayed a reduced pH and thermal stability. In summary we infer that variant influenza viruses with deletions in 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude these viruses would be unlikely to transmit human-to-human by an airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses, as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence.

    IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden, however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as llsquo;antigenic driftrrsquo;. Previously we experimentally generated antigenic drift variants in the laboratory, and here we test our llsquo;driftedrrsquo; viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the llsquo;driftedrrsquo; viruses were able to infect and transmit between chickens and showed increased binding to human-like receptors. However the llsquo;driftrrsquo; mutant viruses displayed reduced stability and, we predict, would be unlikely to be able to transmit human-to-human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and potential of avian influenza viruses to infect humans.

  • Antibody-induced internalization of the human respiratory syncytial virus fusion protein [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigennndash;antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (pAbs) or RSV glycoprotein-specific monoclonal antibodies (mAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs) and RSV isolates suggest that antibody-internalization can be considered as a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin-dependent. All RSV F-targeted mAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different mAbs, indicating that RSV F internalization was epitope-independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.

    IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigennndash;antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms towards infected cells.

  • Transmission Bottleneck Size Estimation from Pathogen Deep-Sequencing Data, with an Application to Human Influenza A Virus [PublishAheadOfPrint]

  • The bottleneck governing infectious disease transmission describes the size of the pathogen population transferred from donor to recipient host. Accurate quantification of the bottleneck size is particularly important for rapidly evolving pathogens such as influenza virus, as narrow bottlenecks reduce the amount of transferred viral genetic diversity and, thus, may slow the rate of viral adaptation. Previous studies have estimated bottleneck sizes governing viral transmission using statistical analyses of variants identified in pathogen sequencing data. These analyses, however, did not account for variant calling thresholds and stochastic viral replication dynamics within recipient hosts. Because these factors can skew bottleneck size estimates, we introduce a new method for inferring bottleneck sizes that accounts for these factors. Through the use of a simulated dataset, we first show that our method, based on beta-binomial sampling, accurately recovers transmission bottleneck sizes, whereas other methods fail to do so. We then apply our method to a dataset of influenza A infections for which viral deep-sequencing data from transmission pairs are available. We find that the IAV transmission bottleneck size estimates in this study are highly variable across transmission pairs, while the mean bottleneck size of 196 virions is consistent with the previous estimate for this dataset. Further, regression analysis shows a positive association between estimated bottleneck size and donor infection severity, as measured by temperature. These results support findings from experimental transmission studies showing that bottleneck sizes across transmission events can be variable and in part influenced by epidemiological factors.

    IMPORTANCE The transmission bottleneck size describes the size of the pathogen population transferred from donor to recipient host and may affect the rate of pathogen adaptation within host populations. Recent advances in sequencing technology have enabled bottleneck size estimation from pathogen genetic data, though there is not yet a consistency in the statistical methods used. Here, we introduce a new approach to infer the bottleneck size that accounts for variant identification protocols and noise during pathogen replication. We show that failing to account for these factors leads to underestimation of bottleneck sizes. We apply this method to an existing dataset of human influenza infections, showing that transmission is governed by a loose, but highly variable, transmission bottleneck whose size is positively associated with donor infection severity. Beyond advancing our understanding of influenza transmission, we hope this work will provide a standardized statistical approach for bottleneck size estimation for viral pathogens.

  • Selective activation of interferon-{gamma} signaling by Zika virus NS5 protein [PublishAheadOfPrint]

  • Severe complications of Zika virus (ZIKV) infection might be caused by inflammation. How ZIKV induces pro-inflammatory cytokines is not understood. In this study, we show opposite regulatory effect of ZIKV NS5 protein on interferon (IFN) signaling. Whereas ZIKV and its NS5 protein were potent suppressors of type I and type III IFN signaling, they were found to activate IFN- signaling. Inversely, IFN- augmented ZIKV replication. NS5 interacted with STAT2 and targeted it for ubiquitination and degradation, but had no influence on STAT1 stability or nuclear translocation. The recruitment of STAT1-STAT2-IRF9 to IFN-bbeta;-stimulated genes was compromised when NS5 was expressed. Concurrently, the formation of STAT1-STAT1 homodimers and their recruitment to IFN--stimulated genes such as pro-inflammatory cytokine CXCL10 were augmented. Silencing the expression of an IFN- receptor subunit or treatment of ZIKV-infected cells with a JAK2 inhibitor suppressed viral replication and viral induction of IFN--stimulated genes. Taken together, our findings provide a new mechanism by which ZIKV NS5 protein differentially regulates IFN signaling to facilitate viral replication and to cause diseases. This activity might be shared by a group of viral IFN modulators.

    IMPORTANCE Mammalian cells produce three types of interferons to combat viral infection and to control host immune response. To replicate and to cause diseases, pathogenic viruses have developed different strategies to defeat the action of host interferons. Many viral proteins including Zika virus (ZIKV) NS5 protein are known to be able to suppress the antiviral property of type I and type III interferons. Here we further showed that ZIKV NS5 protein can also boost the activity of type II interferon to induce cellular proteins that promote inflammation. This was mediated by the differential effect of ZIKV NS5 protein on a pair of cellular transcription factors STAT1 and STAT2. NS5 induced the degradation of STAT2 but promoted the formation of STAT1-STAT1 protein complex that activates genes controlled by type II interferon. A drug that specifically inhibits IFN- receptor or STAT1 showed anti-ZIKV effect and might also have anti-inflammatory activity.

  • Mutations in the transmembrane domain and cytoplasmic tail of Hendra virus fusion protein disrupt virus-like particle assembly [PublishAheadOfPrint]

  • Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here, we investigated how F endosomal trafficking impacts HeV assembly. We found that HeV matrix (M) and F proteins each induced particle release when expressed alone, but their co-expression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-alone or F-alone VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupt endocytic trafficking led to failure of F to function with M for VLP assembly. Wt F functioned normally for VLP assembly even when its cleavage was prevented with cathepsin inhibitor, indicating that it is F endocytic trafficking that is important for VLP assembly, and not F proteolytic cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release, but retained the ability to incorporate as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to pre-assemble before arriving at plasma membrane budding sites.

    IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for interplay between glycoprotein trafficking and paramyxovirus assembly.

  • Regulation of Tacaribe Mammarenavirus Translation: Positive 5 and Negative 3 Elements, and Role of Key Cellular Factors. [PublishAheadOfPrint]

  • Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic messenger RNAs bearing a capped 5rrsquo; untranslated region (UTR), and lacking 3rrsquo; poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open-reading frame as well as transcripts bearing modified 5rrsquo; and/or 3rrsquo; UTRs were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5rrsquo; end dramatically increases translation efficiency, and that the viral 5rrsquo; UTR comprises stimulatory signals while the 3rrsquo; UTR, and specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had lesser impact on virus-like mRNA translation, as compared with a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5rrsquo; UTR and a negative modulatory element in the 3rrsquo; UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet unknown non-canonical host factors for a cap-dependent initiation mechanism.

    IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic non-pathogenic NW mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.

  • Kaposi's Sarcoma Herpesvirus Latency-Associated Nuclear Antigen (LANA): Replicating and Shielding Viral DNA during Viral Persistence [PublishAheadOfPrint]

  • Kaposi's sarcoma herpesvirus (KSHV) establishes life-long latency. The viral latency-associated nuclear antigen (LANA) promotes viral persistence by tethering the viral genome to cellular chromosomes and by participating in latent DNA replication. Recently, the structure of the LANA C-terminal DNA binding domain was solved and new cytoplasmic variants of LANA were discovered. We discuss how these findings contribute to our current view of LANA structure, assembly and its role during viral persistence.

  • Collateral damage during dengue virus infection: Making sense of DNA by cGAS [PublishAheadOfPrint]

  • Early sensing of viral components or infection-induced tissue damage, is a prerequisite for the successful control of pathogenic viruses by the host innate immune system. Recent results from our laboratory show how immune cells use the DNA sensing machinery to detect intracellular damage generated early during infection with an RNA virus, namely dengue virus (DENV). Conversely, we found that DENV can efficiently dismantle this sensing mechanism by targeting the cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon gene (STING); two crucial host factors involved in DNA detection and type I interferon (IFN) production. These findings highlight the relevance of the DNA sensing mechanism to detect and control infections by RNA viruses. In this review we will discuss how DENV modulate the innate immune DNA sensing pathway, activated in the context of cellular damage during infection.

  • Salivary blockade protects the lower respiratory tract of mice from lethal influenza [PublishAheadOfPrint]

  • It is possible to model progression of influenza virus from the upper respiratory tract to the lower respiratory tract in the mouse using viral inoculum delivered in a restricted manner to the nose. In this model, infection with the A/Udorn/307/72 (Udorn) strain of virus results ultimately in high viral titres in both the trachea and lungs. In contrast, the A/Puerto Rico/8/34 (PR8) strain causes an infection that is almost entirely limited to the nasal passages. The factors that govern the progression of virus down the respiratory tract are not well understood. Here we show that, while PR8 virus grows to high titres in the nose, an inhibitor present in the saliva, blocks further progression of infection to the trachea and lungs, and renders an otherwise lethal dose of virus completely asymptomatic. In vitro, the salivary inhibitor was capable of potent neutralisation of PR8 virus and an additional twenty strains of Type A virus and two Type B strains that were tested. Exceptions were Udorn virus and the closely related H3N2 strains A/Port Chalmers/1/73 and A/Victoria/3/75. Characterisation of the salivary inhibitor showed it to be independent of sialic acid and other carbohydrates for its function. This and other biochemical properties, together with its virus-strain specificity and in vivo function indicate that the mouse salivary inhibitor is a previously undescribed innate inhibitory molecule that may have evolved to provide pulmonary protection of this species from fatal influenza infection.

    IMPORTANCE Influenza A virus occasionally jumps from aquatic birds, its natural host, into mammals to cause outbreaks of varying severity, including pandemics in humans. Despite the laboratory mouse being used as a model to study influenza pathogenesis, natural outbreaks of influenza have not been reported in this species. Here we shed light on one mechanism that might allow mice to be protected from influenza in the wild. We show that virus deposited in the mouse upper-respiratory tract will not progress to the lower-respiratory tract due to the presence of a potent inhibitor of the virus in saliva. Containing inhibitor-sensitive virus to the upper-respiratory tract renders an otherwise lethal infection subclinical. This knowledge sheds light on how natural inhibitors may have evolved to improve survival in this species.

  • Non-structural protein L* species-specificity supports a mouse origin for Vilyuisk human encephalitis virus. [PublishAheadOfPrint]

  • Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is from human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the non-structural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. Using co-immunoprecipitation and functional RNase L assays, the species-specificity of RNase L antagonism was tested for L* from a mouse (DA) and a rat (RTV-1) TMEV strain as well as for VHEV. Co-immunoprecipitation and functional assay data confirmed the species-specificity of L* activity and showed that L* from the rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV.

    IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV) infecting mouse and rat, and Saffold virus (SAFV) infecting human. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), was however isolated from mice that were inoculated with cerebro-spinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV derived from the human sample or from the inoculated mouse. L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species-specificity even allows discriminating between TMEV strains of mouse and of rat origin. VHEV L* protein clearly inhibited mouse but not human RNase L, indicating that this virus originates from the mouse.

  • Cellular cholesterol facilitates the post-entry replication cycle of herpes simplex virus 1 [PublishAheadOfPrint]

  • Cholesterol is an essential component of cell membranes and is required for HSV-1 entry (1-3). Treatment of HSV-1-infected Vero cells with methyl beta-cyclodextrin from 2 nndash; 9 hours post-entry reduced plaque numbers. Transport of incoming viral capsids to the nuclear periphery was unaffected by cholesterol reduction suggesting that cell cholesterol is important for the HSV-1 replicative cycle at a stage(s) beyond entry after the arrival of capsids at the nucleus. The synthesis and release of infectious HSV-1 and cell-to-cell spread of infection were all impaired in cholesterol-reduced cells. Propagation of HSV-1 on DHCR24-/- fibroblasts, which lack the desmosterol-to-cholesterol conversion enzyme, resulted in the generation of infectious, extracellular virions (HSVdes) that lack cholesterol and likely contain desmosterol. The specific infectivity (PFU per viral genome) of HSVchol and HSVdes were similar suggesting cholesterol or desmosterol in the HSV envelope support similar levels of infectivity. However, infected DHCR24-/- fibroblasts released ~1 log less infectious HSVdes and ~1.5 logs fewer particles compared to release of cholesterol-containing particles (HSVchol) from parental fibroblasts, suggesting that the hydrocarbon tail of cholesterol facilitates viral synthesis. Together, the results suggest multiple roles for cholesterol in the HSV-1 replicative cycle.

    Importance HSV-1 infections are associated with a wide range of clinical manifestations that are of public health importance. Cholesterol is a key player in the complex interaction between viral and cellular factors that allows HSV-1 to enter host cells and establish infection. Previous reports have demonstrated a role for cellular cholesterol in the entry of HSV-1 into target cells. Here, we employ both chemical treatment and cells that are genetically defined to synthesize only desmosterol to demonstrate that cholesterol is important at stages following the initial entry and transport of viral capsids to the nucleus. Viral protein expression, encapsidation of viral genome and the release of mature virions were impacted by the reduction of cellular cholesterol. Cholesterol was also critical for cell-to-cell spread of infection. These findings provide new insights into the cholesterol-dependence of HSV-1 replication.

  • Minute Virus of Mice (MVM) inhibits transcription of the cyclin B1 gene during infection [PublishAheadOfPrint]

  • Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent pre-mitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others previously reported, depends upon the significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected, the production of nascent cyclin B1 RNA was substantially diminished at late times post infection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein and RNA normally increased in response to DNA damaging reagents. We demonstrate that at times during infection of reduced cyclin B1 expression there was significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 promoter. Additionally, while total Fox M1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 or the activation domain of FoxM1 to the cyclin B1 promoter via enzymatically inactive CRISPR-Cas9 in MVM infected cells increased both cyclin B1 protein and RNA, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection.

    IMPORTANCE Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus exploits to prepare the nuclear environment for effective takeover. An essential aspect of the MVM-induced DDR is establishment of a potent pre-mitotic block. This block depends upon the significant, specific depletion of cyclin B1 and its encoding RNA that precludes cyclin B1/CDK1 complex functions necessary for mitotic entry. We show that reduced cyclin B1 expression was controlled primarily at the level of transcription initiation. Additionally, the essential mitotic transcription factor FoxM1 and RNA polymerase II were found to occupy the cyclin B1 promoter at reduced levels during infection. Recruiting a constitutively active FoxM1, or the activation domain of FoxM1, to the cyclin B1 promoter via CRISPR-dCas9 in MVM infected cells increased expression of both cyclin B1 protein and RNA, implicating FoxM1 as a critical target mediating MVM-induced cyclin B1 inhibition.

  • Dense array of spikes on HIV-1 virion particles [PublishAheadOfPrint]

  • HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e. ~7-14. This exceptional feature has been associated with avoidance of humoral immunity, i.e. B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence activated cell sorting (FACS) to sort for cells that are recognized by trimer crossreactive broadly neutralizing antibody (bnAb) and not by non-neutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (p = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and display more than a ten-fold increase in spikes per particle by electron microscopy (average: 127 spikes; range: 90-214). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of llsquo;cell factoryrrsquo; selection were required for the high-Env phenotype. Relative to standard pseudovirions hVLPs showed greater infectivity but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.

    IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by non-neutralizing antibodies. Levels of spikes on cells correlated well with that on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens.

  • Grass Carp Reovirus VP41 Targets Fish MITA to Abrogate the IFN Response [PublishAheadOfPrint]

  • Although fish possess an efficient interferon (IFN) system to defend against aquatic virus infection, the grass carp reovirus (GCRV) still causes hemorrhagic disease in grass carp. To date, GCRV's strategy for evading the fish IFN response is still unknown. Here, we report that the GCRV VP41 inhibits fish IFN production by suppressing the phosphorylation of the mediator of IRF3 activation (MITA). First, the activation of the IFN promoter (IFNpro) stimulated by the mitochondrial antiviral signaling protein (MAVS) and MITA was decreased by the overexpression of VP41, whereas such activation induced by TANK-binding kinase 1 (TBK1) was not affected. Second, VP41 co-localized in the cellular endoplasmic reticulum (ER) and associated with MITA. Furthermore, as a phosphorylation substrate of TBK1, VP41 significantly decreased the phosphorylation of MITA. Truncation assays indicated that the transmembrane (TM) region of VP41 was indispensable to the suppression of IFNpro activity. Finally, after infection with GCRV, VP41 blunted the transcription of host IFN and facilitated viral RNA synthesis. Taken together, our findings suggest that the GCRV VP41 prevents the fish IFN response by attenuating the phosphorylation of MITA for viral evasion.

    IMPORTANCE MITA is thought to act as an adaptor protein to facilitate the phosphorylation of IRF3 by TBK1 upon viral infection, and it plays a critical role in innate antiviral responses. Here, we report that the GCRV VP41 co-localizes with MITA at the ER, and reduces MITA phosphorylation by acting as a decoy substrate of TBK1, thus inhibiting IFN production. These findings reveal GCRV's strategy for evading the host IFN response for the first time.

  • Analysis of competing HIV-1 splice donor sites uncovers a tight cluster of splicing regulatory elements within exon 2/2b [PublishAheadOfPrint]

  • The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Non-coding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For the vif mRNA, intron 1 must be removed, while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented.

    Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion into viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impact of these SREs on the viral splicing pattern and protein expression was exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by usage of locked nucleic acids (LNAs) impaired Vif expression and viral replication.

    Importance Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNP proteins binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

  • Variable inhibition of Zika virus replication by different Wolbachia strains in mosquito cell cultures [PublishAheadOfPrint]

  • Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with bacterial endosymbiont, Wolbachia pipientis wMel, is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel potentially weakening the integration of this strain into a mosquito population relative to other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. The Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.

    IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the western hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia. Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.

  • Differential involvement during latent HSV-1 infection of the superior and inferior divisions of the vestibular ganglia: implications for vestibular neuritis [PublishAheadOfPrint]

  • Controversy still surrounds both the etiology and pathophysiology of vestibular neuritis (VN). Especially uncertain is why the superior vestibular nerve (SVN) is more frequently affected than the inferior part (IVN), which is partially or totally spared. To address this question we developed an improved method for preparing human vestibular ganglia (VG) and nerve. Subsequently macro- and micro-anatomical as well as PCR studies were performed on 38 human ganglia from 38 individuals. The SVN was 2.4 mm longer than the IVN, and in 65% of the cases the IVN ran in two separate bony canals, which was not the case for the SVN. Anastomoses between the facial and cochlear nerves were more common to the SVN (14/38 and 9/38, respectively) than to the IVN (7/38 and 2/38, respectively). Using single-cell RT-qPCR we found only a few HSV-1 latently infected VG (18.4%). In cases of two separate neuronal fields infected neurons were located in the superior part only. In summary, these PCRs, micro-, and macro-anatomical studies provide possible explanations for the high frequency of SVN infection in vestibular neuritis.

    IMPORTANCE Vestibular neuritis is known to affect the superior part of the vestibular nerve more frequently than the inferior part. The reason for this clinical phenomenon remains unclear. Anatomical differences may play a role or if a latent HSV-1 infection is assumed, the etiology may be due to the different distribution of the infection. To shed further light on this subject, we conducted different macro- and micro-anatomical studies. We also assessed the presence of HSV-1 in VG and in different sections of the VG. Our findings add new information on the macro- and microanatomy of the VG as well as the pathophysiology of vestibular neuritis. We also show that latent HSV-1 infection of the VG neurons is less frequent than previously reported.

  • Disparate contributions of human retrovirus capsid subdomains to Gag-Gag oligomerization, virus morphology and particle biogenesis [PublishAheadOfPrint]

  • The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside of CA (e.g. matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology.

    IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the capsid carboxy terminal domain (CA CTD) is the primary determinant of Gag-Gag interactions for human immunodeficiency virus type 1 HIV-1. In this study, the human T-cell leukemia virus type 1 (HTLV-1) capsid amino terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.

  • Comparative genomics of Chrysochromulina Ericina Virus (CeV) and other microalgae-infecting large DNA viruses highlight their intricate evolutionary relationship with the established Mimiviridae family [PublishAheadOfPrint]

  • Chrysochromulina Ericina Virus CeV-01B (CeV) was isolated from Norwegian coastal waters in 1998. Its icosahedral particle is 160 nm in diameter and encloses a 474-kb dsDNA genome. This virus, although infecting a microalgae (the haptophyceae Haptolina ericina, formerly Chrysochromulina ericina), is phylogenetically related to members of the Mimiviridae family, initially established with the acanthamoeba-infecting Mimivirus and Megavirus as prototypes. This family was latter split into two genera (Mimivirus and Cafetariavirus) following the characterization of a virus infecting the heterotrophic stramenopile Cafeteria roenbergensis (CroV). CeV, as well as two of its close relatives infecting the unicellular photosynthetic eukaryotes Phaeocystis globosa (PgV) and Aureococcus anophagefferens (AaV), are currently unclassified by ICTV. The detailed comparative analysis of the CeV genome presented here confirms the phylogenetic affinity of this emerging group of microalgae-infecting viruses with the Mimiviridae, but argues in favor of their classification inside a distinct clade within the family. Although CeV, PgV, AaV share more common features between them than with the larger Mimiviridae, they also exhibit a large complement of unique genes attesting to their complex evolutionary history. We identified several gene fusion events and cases of convergent evolution involving independent lateral gene acquisitions. Finally, CeV possesses an unusual number of inteins, some of which are closely related despite been inserted in non-homologous genes. This appears to contradict the paradigm of allele-specific inteins and suggests that Mimiviridae might be especially efficient in spreading inteins while enlarging their repertoire of homing genes.

    Importance Although it infects the microalgae Chrysochromulina ericina, CeV is more closely related to acanthamoeba-infecting viruses of the Mimiviridae family than to any member of the Phycodnaviridae, the ICTV-approved family historically including all algae-infecting large dsDNA viruses. CeV, as well as its relatives infecting the other microalgae Phaeocystic globosa (PgV) and Aureococcus anophagefferens (AaV), remain officially unclassified and a source of confusion in the literature. Our comparative analysis of the CeV genome in the context of this emerging group of algae-infecting viruses suggests that they belong to a distinct clade within the established Mimiviridae family. The presence of a large number of unique genes as well as specific gene fusion events, evolutionary convergences, and inteins integrated at unusual locations document the complex evolutionary history of the CeV lineage.

  • Pacmanvirus, a new giant icosahedral virus at the crossroads between Asfarviridae and Faustoviruses. [PublishAheadOfPrint]

  • African swine fever virus, a double stranded DNA virus infecting pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of Faustovirus followed by the description of Kaumoebavirus, carried out over the past two years, we observed the emergence of previously unknown related viruses within this group of viruses. Here, we describe the isolation of Pacmanvirus, a fourth member in this group, capable of infecting Acanthamoeba castellanii. Pacmanvirus A23 has a linear compact genome of 395,405 base pairs in length with a 33.62 G+C content. The Pacmanvirus genome harbors 465 genes with a high coding density. The analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, Pacmanvirus, Faustovirus and Kaumoebavirus. Moreover, the major capsid protein locus of Pacmanvirus appears to be different when compared to those of Kaumoebavirus and Faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, Faustovirus, Pacmanvirus, and Kaumoebavirus.

    Importance Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus isolated from an environmental sample by amoeba co-culture. We describe herein its structure and replicative cycle, along with the genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus after Faustovirus and Kaumoebavirus that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.

  • Serine 235 Is the Primary NS5A Hyper-Phosphorylation Site Responsible for HCV Replication [PublishAheadOfPrint]

  • The non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyper-phosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyper-phosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235 phosphorylated NS5A constituted the primary hyper-phosphorylated NS5A species. S235A mutation blunted viral replication whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyper-phosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase I aalpha; that requires a priming phosphorylation at S235.

    IMPORTANCE It has been known for years that the hepatitis C virus non-structural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyper-phosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyper-phosphorylation and functions; however, the primary serine residue responsible for NS5A hyper-phosphorylation is not clear. Here, we show for the first time that serine 235 phosphorylated NS5A constitutes the primary hyper-phosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner i.e. phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.

  • Mouse saliva inhibits transit of influenza virus to the lower respiratory tract by efficiently blocking influenza neuraminidase activity [PublishAheadOfPrint]

  • We have previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix proteins, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequence of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368-370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location and mutation to either EES or KDS found in highly susceptible strains significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surface of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function though not mechanism to what man has created synthetically as an antiviral drug for influenza.

    IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza infection and pathogenesis, to our knowledge mice do not naturally succumb to influenza. Here we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops virus from reaching the lungs, providing a possible mechanism for why this species may not experience severe influenza infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection.

  • Broadening CD4+ and CD8+ T cell responses against hepatitis C virus by vaccination with NS3 overlapping peptide panels in cross-priming liposomes [PublishAheadOfPrint]

  • Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employ a vaccine approach based on a mix of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09), to facilitate a versatile presentation of all possible T cell epitopes, regardless of HLA-background of the vaccine recipient. Here, we demonstrated that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 only induced CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells such as IFN-+TNF-aalpha;+ co-producers and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV inducing a broadened T cell response targeting both immunodominant- and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and preventing chronicity.

    Importance: With at least 700,000 annual deaths development of a vaccine against hepatitis C virus (HCV) is of high priority, but the tremendous ability of this virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV, have a remarkable ability to drive initially strong CD4+ and CD8+ T cell responses against dominant epitopes towards an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine-strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof-of-concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional and cytotoxic CD4+ and CD8+ T cells when compared to vaccination with rNS3, which generated CD4+ T cell responses only.

  • A novel type of polyhedral viruses infecting hyperthermophilic archaea [PublishAheadOfPrint]

  • Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known virus. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20 kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales. The SPV1 genome consists of 20,222 bp and encodes 45 open reading frames, only one fifth of which could be functionally annotated.

    IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not explored employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered as a representative of a new viral family.

  • Distinct roles of vaccinia virus NF-kB inhibitor proteins A52, B15 and K7 in the immune response [PublishAheadOfPrint]

  • Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-B pathway inhibitors A52, B15 and K7 in immunity, we deleted their genes in a NYVAC vaccinia virus strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of A52R, B15R and K7R increased dendritic cell, natural killer cell and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses for these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double- and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly.

    IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of central host-cell signaling pathway. In mice we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design.

  • Disruption of MDA5 mediated innate immune responses by the 3C proteins of Coxsackievirus A16, Coxsackievirus A6, and Enterovirus D68 [PublishAheadOfPrint]

  • Coxsackievirus A16 (CV-A16), A6 (CV-A6), and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3Cpro proteins from CV-A16, CV-A6, and EV-D68 bind MDA5 and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I IFN signaling in the RLR pathway was blocked by CV-A16, CV-A6, and EV-D68 3Cpro. Furthermore, CV-A16, CV-A6, and EV-D68 3Cpro all cleave TAK1, resulting in inhibition of NF-B activation, a host response also critical for toll-like receptor (TLR) mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3Cpro from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN anti-viral function, offering a potential novel anti-viral strategy.

    IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3Cpro protease blocks MDA5-triggered type I IFN induction. 3Cpro of these viruses binds MDA5 and inhibits its interaction with MAVS. In addition, CV-A16, CV-A6, and EV-D68 3Cpro cleaves TAK1 to inhibit the NF-B response. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting CV-A16, CV-A6, and EV-D68 3Cpro as an anti-viral strategy.

  • Formation of a viral replication focus in Sulfolobus cells infected by the rudivirus SIRV2 [PublishAheadOfPrint]

  • Viral factories are compartmentalized centres for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by the prototypical archaeal virus SIRV2 in the model archaeon Sulfolobus. This rod-shaped virus belongs to the viral family rudiviridae, carrying linear dsDNA genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that only Dpo1 of the four host DNA polymerases (Dpo1 to Dpo4) participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that the organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life.

    IMPORTANCE The organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication centre by a virus infecting members of the Archaea domain.

  • Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets [PublishAheadOfPrint]

  • Glycosylation of Env defines pathogenic properties of SIV. We previously demonstrated that pathogenic SIVmac239 and live-attenuated, quintuple deglycosylated Env mutant (5G) target CD4+ T cells residing in different tissues during acute infection. SIVmac239 and 5G preferentially infected distinct CD4+ T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012). Herein, we studied the host responses relevant to SIV targeting of CXCR3+CCR5+ CD4+ T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with a robust expression of CXCL10, CXCR3+ T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs were secondary to the induction of CD14+CD16+ monocytes and MAC387+ macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387+ macrophages, T cells, dendritic cells (DCs) and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239-infection. Restricted infection in SLOs by 5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DCs lineage.

    IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 and a live-attenuated, deglycosylated mutant 5G infected distinct CD4+ T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012). Accordingly, infections with SIVmac239, but not with 5G, deplete CXCR3+CCR5+CD4+ T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with 2 different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory-Th1 responses than 5G. In particular, SIVmac239 infection elicited a robust expression of CXCL10, a chemokine for CXCR3+ cells, in CD14+CD16+ monocytes and MAC387+ macrophages, recently infiltrated in SLOs. In contrast, 5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets, and elicits marked differences in SIV infection and clinical outcomes.

  • Equine Arteritis Virus has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but not Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract [PublishAheadOfPrint]

  • Equine arteritis virus (EAV) has a global impact on the equine industry being the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is only detectable in the reproductive tract and viral persistence occurs despite the presence of high serum neutralizing antibodies. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host-cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e. testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes, and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages and dendritic cells (Iba-1+ and CD83+), with a low number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation.

    Importance: The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary site of viral persistence combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages), T and B lymphocytes in the presence of detectable inflammatory responses suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

  • Protective efficacy of formaldehyde-inactivated whole-virus vaccine and antivirals in a murine model of coxsackievirus A10 infection [PublishAheadOfPrint]

  • Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, foot and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, which can be complicated by severe neurological sequelae. Morbidity and mortality of CVA10-associated HFMD has been increasing in recent years, particularly in the pan-Pacific region. There are limited studies however on the pathogenesis and immunology of CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly five-day-old ICR mice, which developed significant clinical signs including: reduced mobility, lower weight gain and quadriplegia, with significant pathology in the brain, hind limb skeletal muscles and lungs of infected mice in the moribund state. The severity of illness was associated with abnormally high expression of the proinflammatory cytokine IL-6. Antiviral assays demonstrated that ribavirin and gamma interferon administration could significantly inhibit CVA10 replication both in vitro and in vivo. In addition, formaldehyde-inactivated CVA10 whole-virus vaccines induced immune responses in adult mice and maternal neutralizing antibodies could be transmitted to neonatal mice providing protection against CVA10 clinical strains. Furthermore, high titer antisera were effective against CVA10 and could relieve early clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In summary, we present a novel murine model to study CVA10 pathology which will be extremely useful to develop effective antivirals and vaccines to diminish the burden of HFMD-associated disease.

    IMPORTANCE Hand, foot and mouth disease cases in infancy, arising from coxsackievirus A10 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe disease and fatalities can however occur in some children, necessitating the development of vaccines and antiviral therapies. The present study has established a newborn mouse model of CVA10 which, importantly, recapitulates many aspects of human disease, with respect to the neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon could decrease viral titers in vitro and in vivo. Whole-virus vaccines produced immune responses in adult mice and immunized mothers conferred protection to neonates against challenge from CVA10 clinical strains. Passive immunization with high titer antisera could also improve survival rates in newborn animals.

  • Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity are Consistent with Inter-Subtype Differences in HIV-1 Disease Progression [PublishAheadOfPrint]

  • There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates (r=0.51; p=0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterised cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases (pllt;0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as a major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities: subtypes A/C llt; D llt; inter-subtype recombinants (pllt;0.0029), which is consistent with reported inter-subtype differences in disease progression. We thus hypothesise that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes.

    IMPORTANCE HIV-1 subtypes are unevenly distributed globally and there are reported differences in rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that HIV-1 Gag-protease-driven replication capacity correlates with the replication capacity of whole virus isolates. We further show that subtype B displays significantly higher Gag-protease-mediated replication capacity compared to subtype C, and we identify a major genetic determinant of these differences. Moreover, in two independent East African cohorts we demonstrate a reproducible hierarchy of Gag-protease-driven replicative capacity where recombinants exhibit the greatest replication, followed by subtype D, followed by subtype A and C. Our data identify Gag-protease as a major determinant of subtype differences in disease progression among HIV-1 subtypes; furthermore we propose that the poorer viral replicative capacity of subtypes A and C may paradoxically contribute to their more efficient spread in Sub-Saharan Africa.

  • The major envelope glycoprotein of Murid Herpesvirus-4 promotes sexual transmission [PublishAheadOfPrint]

  • Gammaherpesviruses are important human and animal pathogens. Infection control has proved difficult because the key process of transmission is ill-understood. Murid herpesvirus-4 (MuHV-4), a gammaherpesvirus of mice, transmits sexually. We show that this depends on the major virion envelope glycoprotein, gp150. Gp150 is redundant for host entry and, in vitro, it regulates rather than promotes cell binding. We show that gp150-deficient MuHV-4 reaches and replicates normally in the female genital tract after nasal infection, but is poorly released from vaginal epithelial cells and fails to pass from the female to the male genital tract during sexual contact. Thus, we show that regulation of virion binding is a key component of spontaneous gammaherpesvirus transmission.

    IMPORTANCE Gammaherpesviruses are responsible for many important diseases both in animals and humans. Some important aspects of their lifecycle are still poorly understood. Key amongst these is viral transmission. Here we show that the major envelope glycoprotein of Murid Herpesvirus 4 functions not in entry or dissemination, but in virion release to allow sexual transmission to new hosts.

  • Virion associated Vpr alleviates a post-integration block to HIV-1 infection of dendritic cells [PublishAheadOfPrint]

  • Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr in facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cells to encounter virus in the peripheral mucosal tissues. We characterize in this report a significant restriction to Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC mmdash; CD4+ T cell co-cultures. This restriction to HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants either unable to interact with DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1 infected MDDCs was unaffected by the absence of Vpr, transcriptional activity of the viral LTR from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel post-integration restriction to HIV-1 replication in MDDCs and that Vpr interaction with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement to HIV-1 replication.

    IMPORTANCE Despite decades of work, function of the HIV-1 protein Vpr remains poorly understood, primarily due to lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

  • Phosphorylation induces structural changes in the Autographa californica nucleopolyhedrovirus P10 protein [PublishAheadOfPrint]

  • Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10 kDa auxiliary protein produced in the very-late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeletal-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated peri-nuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate horizontal transmission of the virus between insect hosts. Here it is demonstrated, using mass spectrometric analysis, that the C-terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of the P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed an aberrant formation of the peri-nuclear tubules. Thus, phosphorylation of serine 93 may induce aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects P10 structural conformation.

    IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during the viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for survival of its progeny and therefore P10 presents an enigma. As P10 polymerises to form organised cytoskeletal structures that co-localise with the host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus, potentially, be responsible for its dissemination and survival.

  • Characterization of Pre-F-GCN4t, a modified human respiratory syncytial virus fusion protein stabilized in a non-cleaved pre-fusion conformation [PublishAheadOfPrint]

  • Human respiratory syncytial virus (hRSV) fusion protein (F) is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from pre- (pre-F) to post-fusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state may be a superior target for neutralizing antibodies than post-F. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated, trimeric, and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical-shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to more efficiently deplete hRSV neutralizing antibodies from human serum, compared with Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to post-F conformation and present antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered as a promising candidate vaccine antigen.

    IMPORTANCE Human Respiratory Syncytial Virus (RSV) is a global leading cause of infant mortality and adult morbidity. Development of a safe and efficacious RSV vaccine remains an important goal. RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates and recent evidences suggest that the pre-fusion state (pre-F) might be a superior target for neutralizing antibodies. Our study presents the physico-chemical characterization of Pre-F-GCN4t, a molecule meant to be stabilized in pre-F conformation. To confirm its stabilization in a pre-F state, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F molecule and support its use as an RSV vaccine antigen. Such antigen may represent a significant advance in the development of an RSV vaccine.

  • Stress Hormones Epinephrine and Corticosterone Selectively Modulate HSV-1 and HSV-2 Productive Infection in Adult Sympathetic, but not Sensory, Neurons [PublishAheadOfPrint]

  • Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with exacerbation of clinical symptoms and induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infection within sensory and autonomic neurons, we analyzed viral DNA and production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and glucocorticoid receptor (activated by corticosterone). Productive HSV infection co-localized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased HSV-2 DNA replication and production of viral progeny in SCG neurons, but not in TG neurons. Thus, stress-related hormones, epinephrine and corticosterone, selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons.

    IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become infected by HSV-1 and HSV-2 express stress hormone receptors and are responsive to hormone fluctuations. Our results show that autonomic neurons are more responsive to epinephrine and corticosterone than are sensory neurons, demonstrating that the autonomic nervous system plays a substantial role in HSV pathogenesis. Furthermore, these results suggest that stress responses have the potential to differentially impact HSV-1 and HSV-2 to produce divergent outcomes of infection.

  • Genome-Wide Screening Uncovers the Significance of N-sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection [PublishAheadOfPrint]

  • The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3mmdash;E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of a clinically isolated CHIKV Thai #16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). Whereas glycosaminoglycans were commonly required for efficient infectivity of CHIKV, JEV and YFV as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at the later steps after the CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection, because it is involved in proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism and pathogenesis.

    IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. Comparing previous reports that used Chinese hamster ovary cells, these findings together with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells alert that infection manner of CHIKV varies among the host cells. We also reveal that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.

  • Attachment and Post-Attachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection requires multiple receptors for its attachment and entry to the cell. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules such as CD81, CLDN1, OCLN, SR-BI, and LDLR mainly function at post-attachment steps, which are considered post-attachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a CRISPR/Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockout of SDC-1 and SDC-2 resulted in a remarkable reduction of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockout did not affect HCV infection. The defective HCV attachment in the SDC-1 and/or SDC-2 knockout cells could be completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the post-attachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread but the VLDL-containing mouse serum did not affect HCV cell-to-cell transmission although it inhibited its cell-free infection. These findings demonstrate that attachment receptors are essential for the initial HCV binding and post-attachment receptors are important for both HCV cell-free and cell-to-cell transmission.

    IMPORTANCE The importance and underlying molecular mechanisms of cell surface receptors in HCV cell-free and cell-to-cell transmission are poorly understood. The role of some of the HCV attachment and post-attachment receptors in HCV infection and cell-to-cell spread remains controversial. Using CRISPR/Cas9-mediated knockout of specific cellular genes, we demonstrate that both SDC-1 and SDC-2 but not SDC-3 or SDC-4 are bona fide HCV attachment receptors. We also used a newly developed luciferase-based reporter system to quantitatively determine the importance of attachment and post-attachment receptors in HCV cell-to-cell transmission. SDC-1, SDC-2, TIM-1, and SR-BI were found to modestly promote HCV cell-to-cell spread. CD81, CLDN1, OCLN, and LDLR play more important roles in HCV cell-to-cell transmission. Likewise, apoE is critically important for HCV cell-to-cell spread unlike the VLDL-containing mouse serum that did not affect HCV cell-to-cell spread. These findings suggest that the mechanism(s) of HCV cell-to-cell spread differ from that of its cell-free infection.

  • Membrane penetration by bacterial viruses [PublishAheadOfPrint]

  • The bacteriophage 29 infects gram-positive Bacillus subtilis with a short non-contractile tail. Recent studies have shown that the 29 tail protein gp9 forms a hexameric tube with six long loops of membrane-active peptides blocking in the tube at the distal end of the tail. The long loops exit upon genome release and form a membrane pore for passage of the genome. The membrane penetration mechanism of the 29 tail might be common among tailed bacteriophages.

  • Herpes virus entry mediator (HVEM) and ocular herpes infection: more than meets the eye [PublishAheadOfPrint]

  • As its name suggests, the host receptor herpes virus entry mediator (HVEM) facilitates herpes simplex virus (HSV) entry through interactions with a viral envelope glycoprotein. HVEM also bridges several signaling networks, binding ligands from both tumor necrosis factor (TNF) and immunoglobulin (Ig) superfamilies with diverse, and often opposing, outcomes. While HVEM was first identified as a viral entry receptor for HSV, it is only recently that HVEM has emerged as an important host factor in immunopathogenesis of ocular HSV type 1 (HSV-1) infection. Surprisingly, HVEM exacerbates disease development in the eye independently of entry. HVEM signaling has been shown to play a variety of roles in modulating immune responses to HSV and other pathogens, and there is increasing evidence that these effects are responsible for HVEM-mediated pathogenesis in the eye. Here, we review the dual branches of HVEM function during HSV infection: entry and immunomodulation. HVEM is broadly expressed, intersects two important immunologic signaling networks, and impacts autoimmunity, infection, and inflammation. We hope that by understanding the complex range of effects mediated by this receptor, we can offer insights applicable to a wide variety of disease states.

  • Autologous stem-cell transplantation disrupts adaptive immune responses during rebound SHIV viremia [PublishAheadOfPrint]

  • Primary HIV-1 infection induces a virus-specific adaptive/cytolytic immune response that impacts plasma viral load setpoint and rate of progression to AIDS. Combination antiretroviral therapy (cART) suppresses plasma viremia to undetectable levels that rebound upon cART treatment interruption. Following cART withdrawal, the memory component of the virus-specific adaptive immune response may improve viral control compared to primary infection. Here, using primary infection and treatment interruption data from macaques infected with simian/human immunodeficiency virus (SHIV), we observe lower peak viral load but unchanged viral setpoint during viral rebound. The addition of an autologous stem-cell transplant before cART withdrawal alters viral dynamics: we find a higher rebound setpoint but similar peak viral loads compared to primary infection. Mathematical modeling of the data that accounts for fundamental immune parameters achieves excellent fit to heterogeneous viral loads. Analysis of model output suggests that the rapid memory immune response following treatment interruption does not ultimately lead to better viral containment. Transplantation decreases the durability of the adaptive immune response following cART withdrawal and viral rebound. Our model's results highlight the impact of the endogenous adaptive immune response during primary SHIV infection. Moreover, because we capture adaptive immune memory and the impact of transplantation, this model will provide insight into further studies of cure strategies inspired by the Berlin patient.

    Importance: HIV patients who interrupt combination antiretroviral therapy (cART) eventually experience viral rebound, the return of viral loads to pre-treatment levels. However, the "Berlin patient" remains free of HIV rebound over a decade after stopping cART. His cure is attributed to leukemia treatment that included an HIV-resistant stem cell transplant. Inspired by this case, we studied the impact of stem-cell transplantation in a macaque simian/HIV (SHIV) system. Using a mechanistic mathematical model, we found that while primary infection generates an adaptive immune memory response, stem cell transplantation disrupts this learned immunity. The results have implications for HIV cure regimens based on stem-cell transplantation.

  • Discovery of Novel Small Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1 [PublishAheadOfPrint]

  • A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity through hijacking chemokine coreceptor signaling which activates a host dependency factor cofilin and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIMK1 with shRNA inhibits HIV infection, no specific small molecule inhibitor of LIMK was available. Here we describe the design and discovery of novel classes of small molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK kinase activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses including EBOV, RVFV, VEEV, and HSV-1, suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum anti-viral drugs.

    IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases such as the LIM domain kinase (LIMK) that regulates actin activity through phosphorylation of cofilin, an actin depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small molecule LIMK inhibitors for blocking HIV-1 and several other viruses. This study emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

  • Impact of antibodies and strain polymorphisms on cytomegalovirus entry and spread in fibroblasts and epithelial cells [PublishAheadOfPrint]

  • Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell-to-cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including gB, gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts formation of syncytia-like structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N-terminus of gH, exhibited strain-specificity, and neutralized both epithelial and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain-specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion, and may assist in determining which antibodies may be most efficacious following active immunization or passive administration.

    IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts; (ii) demonstrate that antibodies can restrict the formation in vitro of syncytia-like structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo; and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain- and cell type-dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion, and may have important implications for the development of CMV vaccines and immunotherapeutics.

  • Pathogenic events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis [PublishAheadOfPrint]

  • Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from107mmdash;109 TCID50 consistently infected all animals, and most monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibility of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), consistent with previous studies of poliovirus pathogenesis in humans. Because this model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, and to assess efficacy of candidate antiviral drugs and new vaccines.

    IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All of 39 macaques fed with single high doses ranging from 107mmdash;109 TCID50 Mahoney type 1 virus were infected, and most monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because this model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

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