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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

  • Editorial Board [Masthead]

  • Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality among immunocompromised and immunonaive individuals. HCMV-induced signaling initiated during viral entry stimulates a rapid noncanonical activation of Akt to drive the differentiation of short-lived monocytes into long-lived macrophages, which is essential for viral dissemination and persistence. We found that HCMV glycoproteins gB and gH directly bind and activate cellular epidermal growth factor receptor (EGFR) and integrin bbeta;1, respectively, to reshape canonical Akt signaling within monocytes. The remodeling of the Akt signaling network was due to the recruitment of nontraditional Akt activators to either the gB- or gH-generated receptor signaling complexes. Phosphoinositide 3-kinase (PI3K) comprised of the p110bbeta; catalytic subunit was recruited to the gB/EGFR complex despite p110 being the primary PI3K isoform found within monocytes. Concomitantly, SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) was recruited to the gH/integrin bbeta;1 complex, which is critical to aberrant Akt activation, as SHIP1 diverts PI3K signaling toward a noncanonical pathway. Although integrin bbeta;1 was required for SHIP1 recruitment, gB-activated EGFR mediated SHIP1 activation, underscoring the importance of the interplay between gB- and gH-mediated signaling to the unique activation of Akt during HCMV infection. Indeed, SHIP1 activation mediated the increased expression of Mcl-1 and HSP27, two Akt-dependent antiapoptotic proteins specifically upregulated during HCMV infection but not during growth factor treatment. Overall, our data indicate that HCMV glycoproteins gB and gH work in concert to initiate an HCMV-specific signalosome responsible for the atypical activation of Akt required for infected monocyte survival and ultimately viral persistence.

    IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic throughout the world regardless of socioeconomic conditions and geographic locations with a seroprevalence reaching up to 100% in some developing countries. Although asymptomatic in healthy individuals, HCMV can cause severe multiorgan disease in immunocompromised or immunonaive patients. HCMV disease is a direct consequence of monocyte-mediated systematic spread of the virus following infection. Because monocytes are short-lived cells, HCMV must subvert the natural short life-span of these blood cells by inducing a distinct activation of Akt, a serine/theonine protein kinase. In this work, we demonstrate that HCMV glycoproteins gB and gH work in tandem to reroute classical host cellular receptor signaling to aberrantly activate Akt and drive survival of infected monocytes. Deciphering how HCMV modulates the cellular pathway to induce monocyte survival is important to develop a new class of anti-HCMV drugs that could target and prevent spread of the virus by eliminating infected monocytes.

  • Cellular Vimentin Interacts with Foot-and-Mouth Disease Virus Nonstructural Protein 3A and Negatively Modulates Viral Replication [Virus-Cell Interactions]

  • Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

  • Butyrate Reprograms Expression of Specific Interferon-Stimulated Genes [Cellular Response to Infection]

  • Butyrate is an abundant metabolite produced by gut microbiota. While butyrate is a known histone deacetylase inhibitor that activates expression of many genes involved in immune system pathways, its effects on virus infections and on the antiviral type I interferon (IFN) response have not been adequately investigated. We found that butyrate increases cellular infection with viruses relevant to human and animal health, including influenza virus, reovirus, HIV-1, human metapneumovirus, and vesicular stomatitis virus. Mechanistically, butyrate suppresses levels of specific antiviral IFN-stimulated gene (ISG) products, such as RIG-I and IFITM3, in human and mouse cells without inhibiting IFN-induced phosphorylation or nuclear translocation of the STAT1 and STAT2 transcription factors. Accordingly, we discovered that although butyrate globally increases baseline expression of more than 800 cellular genes, it strongly represses IFN-induced expression of 60% of ISGs and upregulates 3% of ISGs. Our findings reveal that there are differences in the IFN responsiveness of major subsets of ISGs depending on the presence of butyrate in the cell environment, and overall, they identify a new mechanism by which butyrate influences virus infection of cells.

    IMPORTANCE Butyrate is a lipid produced by intestinal bacteria. Here, we newly show that butyrate reprograms the innate antiviral immune response mediated by type I interferons (IFNs). Many of the antiviral genes induced by type I IFNs are repressed in the presence of butyrate, resulting in increased virus infection and replication. Our research demonstrates that metabolites produced by the gut microbiome, such as butyrate, can have complex effects on cellular physiology, including dampening of an inflammatory innate immune pathway resulting in a proviral cellular environment. Our work further suggests that butyrate could be broadly used as a tool to increase growth of virus stocks for research and for the generation of vaccines.

  • Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses [Genome Replication and Regulation of Viral Gene Expression]

  • The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.

    IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.

  • Differential Pressures of SERINC5 and IFITM3 on HIV-1 Envelope Glycoprotein over the Course of HIV-1 Infection [Virus-Cell Interactions]

  • Infection of human immunodeficiency virus type 1 (HIV-1) is subject to restriction by cellular factors. Serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3) proteins represent two of these restriction factors, which inhibit HIV-1 entry into target cells. Both proteins impede fusion of the viral membrane with the cellular membrane and the formation of a viral fusion pore, and both are countered by the HIV-1 envelope glycoprotein (Env). Given the immense and lasting pressure which Env endures from host adaptive immune responses, it is important to understand whether and how HIV-1 Env is able to maintain the resistance to SERINC5 and IFITM3 throughout the course of infection. We have thus examined a panel of HIV-1 Env clones that were isolated at different stages of viral infectionmmdash;transmission, acute, and chronic. While HIV-1 Env clones from the transmission stage are resistant to both SERINC5 and IFITM3, as infection progresses into the acute and chronic stages, the resistance to IFITM3 but not to SERINC5 is gradually lost. We further discovered a significant correlation between the resistance of HIV-1 Env to soluble CD4 inhibition and the resistance to SERINC5 but not to IFITM3. Interestingly, the miniprotein CD4 mimetic M48U1 sensitizes HIV-1 Env to the inhibition by SERINC5 but not IFITM3. Together, these data indicate that SERINC5 and IFITM3 exert differential inhibitory pressures on HIV-1 Env over different stages of HIV-1 infection and that HIV-1 Env uses varied strategies to resist these two restriction factors.

    IMPORTANCE HIV-1 Env protein is exposed to the inhibition not only by humoral response, but also by host restriction factors, including serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3). This study investigates how HIV-1 envelope glycoprotein (Env) manages to overcome the pressures from all these different host inhibition mechanisms over the long course of viral infection. HIV-1 Env preserves the resistance to SERINC5 but becomes sensitive to IFITM3 when infection progresses into the chronic stage. Our study also supports the possibility of using CD4 mimetic compounds to sensitize HIV-1 Env to the inhibition by SERINC5 as a potential therapeutic strategy.

  • Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract [Pathogenesis and Immunity]

  • The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.

    IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.

  • Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance [Virus-Cell Interactions]

  • Several serine and threonine residues of the papillomavirus early E2 protein have been found to be phosphorylated. In contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one in HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this study, we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the nonphosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither binds the C terminus of Brd4, but in all other aspects the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in vitro assay, while HPV-31 Y102E binds E1 and was able to replicate, albeit at lower levels than wild type. To examine the effect of E2 mutations under more native-like infection conditions, a neomycin-selectable marker was inserted into L1/L2 of the HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days posttransfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines.

    IMPORTANCE Posttranslational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

  • Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.

    IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread.

  • Host-Virus Arms Races Drive Elevated Adaptive Evolution in Viral Receptors [Genetic Diversity and Evolution]

  • Viral receptors are the cell surface proteins that are hijacked by viruses to initialize their infections. Viral receptors are subject to two conflicting directional forces, namely, negative selection due to functional constraints and positive selection due to host-virus arms races. It remains largely obscure whether negative pleiotropy limits the rate of adaptation in viral receptors. Here, we perform evolutionary analyses of 96 viral receptor genes in primates and find that 41 out of 96 viral receptors experienced adaptive evolution. Many positively selected residues in viral receptors are located at the virus-receptor interfaces. Compared with control proteins, viral receptors exhibit significantly elevated rate of adaptation. Further analyses of genetic polymorphisms in human populations reveal signals of positive selection and balancing selection for 53 and 5 viral receptors, respectively. Moreover, we find that 49 viral receptors experienced different selection pressures in different human populations, indicating that viruses represent an important driver of local adaptation in humans. Our findings suggest that diverse viruses, many of which have not been known to infect nonhuman primates, have maintained antagonistic associations with primates for millions of years, and the host-virus conflicts drive accelerated adaptive evolution in viral receptors.

    IMPORTANCE Viruses hijack cellular proteins, termed viral receptors, to assist their entry into host cells. While viral receptors experience negative selection to maintain their normal functions, they also undergo positive selection due to an everlasting evolutionary arms race between viruses and hosts. A complete picture on how viral receptors evolve under two conflicting forces is still lacking. In this study, we systematically analyzed the evolution of 96 viral receptors in primates and human populations. We found around half of viral receptors underwent adaptive evolution and exhibit significantly elevated rates of adaptation compared to control genes in primates. We also found signals of past natural selection for 58 viral receptors in human populations. Interestingly, 49 viral receptors experienced different selection pressures in different human populations, indicating that viruses represent an important driver of local adaptation in humans. Our results suggest that host-virus arms races drive accelerated adaptive evolution in viral receptors.

  • Restoring Herpesvirus Entry Mediator (HVEM) Immune Function in HVEM-/- Mice Rescues Herpes Simplex Virus 1 Latency and Reactivation Independently of Binding to Glycoprotein D [Pathogenesis and Immunity]

  • The immune modulatory protein herpes virus entry mediator (HVEM) is one of several cellular receptors used by herpes simplex virus 1 (HSV-1) for cell entry. HVEM binds to HSV-1 glycoprotein D (gD) but is not necessary for HSV-1 replication in vitro or in vivo. Previously, we showed that although HSV-1 replication was similar in wild-type (WT) control and HVEMnndash;/nndash; mice, HSV-1 does not establish latency or reactivate effectively in mice lacking HVEM, suggesting that HVEM is important for these functions. It is not known whether HVEM immunomodulatory functions contribute to latency and reactivation or whether its binding to gD is necessary. We used HVEMnndash;/nndash; mice to establish three transgenic mouse lines that express either human WT HVEM or human or mouse HVEM with a point mutation that ablates its ability to bind to gD. Here, we show that HVEM immune function, not its ability to bind gD, is required for WT levels of latency and reactivation. We further show that HVEM binding to gD does not affect expression of the HVEM ligands BTLA, CD160, or LIGHT. Interestingly, our results suggest that binding of HVEM to gD may contribute to efficient upregulation of CD8aalpha; but not PD1, TIM-3, CTLA4, or interleukin 2 (IL-2). Together, our results establish that HVEM immune function, not binding to gD, mediates establishment of latency and reactivation.

    IMPORTANCE HSV-1 is a common cause of ocular infections worldwide and a significant cause of preventable blindness. Corneal scarring and blindness are consequences of the immune response induced by repeated reactivation events. Therefore, HSV-1 therapeutic approaches should focus on preventing latency and reactivation. Our data suggest that the immune function of HVEM plays an important role in the HSV-1 latency and reactivation cycle that is independent of HVEM binding to gD.

  • Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals [Vaccines and Antiviral Agents]

  • Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responsesmmdash;in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5' and 3' terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated "rewired" viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signalsmmdash;thereby increasing the NA content on viral particlesmmdash;is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine.

    IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection.

  • Better Viral Control despite Higher CD4+ T Cell Activation during Acute HIV-1 Infection in Zambian Women Is Linked to the Sex Hormone Estradiol [Pathogenesis and Immunity]

  • The influence of biological sex on disease progression in HIV-1-infected individuals has been focused on the chronic stage of infection, but little is known about how sex differences influence acute HIV-1 infection. We observed profound differences in viral load and CD4+ T cell activation from the earliest time points in men and women in a Zambian heterosexual acute infection cohort. Women exhibited a ggt;2-fold higher rate of CD4+ T cell loss despite significantly lower viral loads (VL) than men. The importance of studying acute infection was highlighted by the observation that very early in infection, women exhibited significantly higher levels of CD4+ T cell activation, a difference that was lost over the first 3 years of infection as activation in men increased. In women, activation of CD4+ T cells in the acute phase was significantly correlated with plasma levels of 17bbeta;-estradiol (E2). However, unlike in men, higher CD4+ T cell activation in women was not associated with higher VL. In contrast, a higher E2 level in early infection was associated with lower early and set-point VL in women. We attribute this to an inhibitory effect of estradiol on virus replication, which we were able to observe with relevant transmitted/founder viruses in vitro. Thus, estradiol plays a key role in defining major differences between men and women during early HIV-1 infection by contributing to both viral control and CD4+ T cell loss, an effect that extends into the chronic phase of the disease.

    IMPORTANCE Previous studies have identified sex-specific differences during chronic HIV-1 infection, but little is known about sex differences in the acute phase, or how disparities in the initial response to the virus may affect disease. We demonstrate that restriction of viral load in women begins during acute infection and is maintained into chronic infection. Despite this, women exhibit more rapid CD4+ T cell loss than men. These profound differences are influenced by 17bbeta;-estradiol, which contributes both to T cell activation and to reduced viral replication. Thus, we conclude that estradiol plays a key role in shaping responses to early HIV-1 infection that influence the chronic phase of disease.

  • Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment [Virus-Cell Interactions]

  • The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.

    IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRVrrsquo;s usage being closer to HSV1rrsquo;s usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

  • An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms [Genome Replication and Regulation of Viral Gene Expression]

  • Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template, whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation, while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudorevertants (L420V and L420I) were selected from all independent lineages of L420A, G64Fix L420A, and G64S L420A parental viruses. Ribavirin resistance G64S mutations were selected in two independent lineages, and novel ribavirin resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, and T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L, M392V, K375R, and R376K). L420A revertants and pseudorevertants (L420V and L420I) restored efficient viral RNA recombination, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L, and M392V) and primer grip mutations (K375R and R376K) exhibited divergent RNA recombination, ribavirin sensitivity, and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375, and R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.

    IMPORTANCE Picornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses, and counteracts error catastrophe. Can viruses distinguish between homologous and nonhomologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and nonhomologous RNA templates during sexual RNA replication.

  • ORF4 of the Temperate Archaeal Virus SNJ1 Governs the Lysis-Lysogeny Switch and Superinfection Immunity [Virus-Cell Interactions]

  • Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes.

    IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

  • cis-Acting Sequences and Secondary Structures in Untranslated Regions of Duck Tembusu Virus RNA Are Important for Cap-Independent Translation and Viral Proliferation [Genome Replication and Regulation of Viral Gene Expression]

  • Duck Tembusu virus (DTMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. The positive-strand RNA genomes of flaviviruses are commonly translated in a cap-dependent manner. However, dengue and Zika viruses also exhibit cap-independent translation. In this study, we show that RNAs containing 5' and 3' untranslated regions (UTRs) of DTMUV, mosquito-borne Tembusu virus (TMUV), and Japanese encephalitis virus can be translated in a cap-independent manner in mammalian, avian, and mosquito cells. The ability of the 5' UTRs of flaviviruses to direct the translation of a second open reading frame in bicistronic RNAs was much less than that observed for internal ribosome entry site (IRES) encephalomyocarditis virus, indicating a lack of substantial IRES activity. Instead, cap-independent translation of DTMUV RNA was dependent on the presence of a 3' UTR, RNA secondary structures located in both UTRs, and specific RNA sequences. Mutations inhibiting cap-independent translation decreased DTMUV proliferation in vitro and delayed, but did not prevent, the death of infected duck embryos. Thus, the 5' and 3' UTRs of DTMUV enable the virus to use a cap- and IRES-independent RNA genome translation strategy that is important for its propagation and virulence.

    IMPORTANCE The genus Flavivirus includes major human pathogens, as well as animal-infecting viruses with zoonotic potential. In order to counteract the threats these viruses represent, it is important to understand their basic biology to develop universal attenuation strategies. Here, we demonstrate that five different flaviviruses use cap-independent translation, indicating that the phenomenon is probably common to all members of the genus. The mechanism used for flavivirus cap-independent translation was found to be different from that of IRES-mediated translation and dependent on both 5' and 3' UTRs that act in cis. As cap-independent translation was also observed in mosquito cells, its role in flavivirus infection is unlikely to be limited to the evasion of consequences of the shutoff of host translation. We found that the inhibition of cap-independent translation results in decreased viral proliferation, indicating that the strategy could be applied to produce attenuated variants of flaviviruses as potential vaccine candidates.

  • An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases [Virus-Cell Interactions]

  • Sirtuin 2 (Sirt2), an NAD+-dependent protein deacetylase, deacetylates tubulin, AKT, and other proteins. Previously, we showed that Sirt2 isoform 1 (Sirt2.1) increased replication of hepatitis B virus (HBV). Here, we show that HBV replication upregulates the expression of Sirt2 primary and alternatively spliced transcripts and their respective isoforms, 1, 2, and 5. Since Sirt2 isoform 5 (Sirt2.5) is a catalytically inactive nuclear protein with a spliced-out nuclear export signal (NES), we speculated that its different localization affects its activity. The overexpression of Sirt2.5 reduced expression of HBV mRNAs, replicative intermediate DNAs, and covalently closed circular DNA (cccDNA), an activity opposite that of Sirt2.1 and Sirt2.2. Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction was weakened by HBV replication. Unlike Sirt2.1, Sirt2.5 activated the AKT/GSK-3bbeta;/bbeta;-catenin signaling pathway very weakly and independently of HBV replication. When the NES and an N-terminal truncated catalytic domain were added to the Sirt2.5 construct, it localized in the cytoplasm and increased HBV replication (like Sirt2.1 and Sirt2.2). Chromatin immunoprecipitation assays revealed that more Sirt2.5 was recruited to cccDNA than Sirt2.1. The recruitment of histone lysine methyltransferases (HKMTs), such as SETDB1, SUV39H1, EZH2, and PR-Set7, and their respective transcriptional repressive markers, H3K9me3, H3K27me3, and H4K20me1, to cccDNA also increased in Sirt2.5-overexpressing cells. Among these, the Sirt2.5nndash;PR-Set7 and nndash;SETDB1 interactions increased upon HBV replication. These results demonstrate that Sirt2.5 reduces cccDNA levels and viral transcription through epigenetic modification of cccDNA via direct and/or indirect association with HKMTs, thereby exhibiting anti-HBV activity.

    IMPORTANCE Sirt2, a predominant cytoplasmic aalpha;-tubulin deacetylase, promotes the growth of hepatocellular carcinoma; indeed, HBV replication increases Sirt2 expression, and overexpression of Sirt2 is associated with hepatic fibrosis and epithelial-to-mesenchymal transition. Increased amounts of Sirt2 isoforms 1, 2, and 5 upon HBV replication might further upregulate HBV replication, leading to a vicious cycle of virus replication/disease progression. However, we show here that catalytically inactive nuclear Sirt2.5 antagonizes the effects of Sirt2.1 and Sirt2.2 on HBV replication, thereby inhibiting cccDNA level, transcription of cccDNA, and subsequent synthesis of replicative intermediate DNA. More Sirt2.5 was recruited to cccDNA than Sirt2.1, thereby increasing epigenetic modification by depositing transcriptional repressive markers, possibly through direct and/or indirect association with histone lysine methyltransferases, such as SETDB1, SUV39H1, EZH2, and/or PR-Set7, which represses HBV transcription. Thus, Sirt2.5 might provide a functional cure for HBV by silencing the transcription of HBV.

  • A Temperature-Dependent Translation Defect Caused by Internal Ribosome Entry Site Mutation Attenuates Foot-and-Mouth Disease Virus: Implications for Rational Vaccine Design [Vaccines and Antiviral Agents]

  • Foot-and-mouth disease (FMD), which is caused by FMD virus (FMDV), remains a major plague among cloven-hoofed animals worldwide, and its outbreak often has disastrous socioeconomic consequences. A live-attenuated FMDV vaccine will greatly facilitate the global control and eradication of FMD, but a safe and effective attenuated FMDV vaccine has not yet been successfully developed. Here, we found that the internal ribosome entry site (IRES) element in the viral genome is a critical virulence determinant of FMDV, and a nucleotide substitution of cytosine (C) for guanine (G) at position 351 of the IRES endows FMDV with temperature-sensitive and attenuation (tsaamp;att) phenotypes. Furthermore, we demonstrated that the C351G mutation of IRES causes a temperature-dependent translation defect by impairing its binding to cellular pyrimidine tract-binding protein (PTB), resulting in the tsaamp;att phenotypes of FMDV. Natural hosts inoculated with viruses carrying the IRES C351G mutation showed no clinical signs, viremia, virus excretion, or viral transmission but still produced a potent neutralizing antibody response that provided complete protection. Importantly, the IRES C351G mutation is a universal determinant of the tsaamp;att phenotypes of different FMDV strains, and the C351G mutant was incapable of reversion to virulence during in vitro and in vivo passages. Collectively, our findings suggested that manipulation of the IRES, especially its C351G mutation, may serve as a feasible strategy to develop live-attenuated FMDV vaccines.

    IMPORTANCE The World Organization for Animal Health has called for global control and eradication of foot-and-mouth disease (FMD), the most economically and socially devastating disease affecting animal husbandry worldwide. Live-attenuated vaccines are considered the most effective strategy for prevention, control, and eradication of infectious diseases due to their capacity to induce potent and long-lasting protective immunity. However, efforts to develop FMD virus (FMDV) live-attenuated vaccines have achieved only limited success. Here, by structure-function study of the FMDV internal ribosome entry site (IRES), we find that the C351 mutation of the IRES confers FMDV with an ideal temperature-sensitive attenuation phenotype by decreasing its interaction with cellular pyrimidine tract-binding protein (PTB) to cause IRES-mediated temperature-dependent translation defects. The temperature-sensitive attenuated strains generated by manipulation of the IRES address the challenges of FMDV attenuation differences among various livestock species and immunogenicity maintenance encountered previously, and this strategy can be applied to other viruses with an IRES to rationally design and develop live-attenuated vaccines.

  • Patterns of Herpes Simplex Virus 1 Infection in Neural Progenitor Cells [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2'-deoxyuridine (5BVdU) and alpha interferon (IFN-aalpha;) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-aalpha;; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1nndash;NPC interaction.

    IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction.

  • Filoviruses Use the HOPS Complex and UVRAG To Traffic to Niemann-Pick C1 Compartments during Viral Entry [Virus-Cell Interactions]

  • Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.

    IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.

  • Aerosol Transmission from Infected Swine to Ferrets of an H3N2 Virus Collected from an Agricultural Fair and Associated with Human Variant Infections [Pathogenesis and Immunity]

  • Influenza A viruses (IAV) sporadically transmit from swine to humans, typically associated with agricultural fairs in the United States. A human seasonal H3 virus from the 2010-2011 IAV season was introduced into the U.S. swine population and termed H3.2010.1 to differentiate it from the previous swine H3 virus. This H3N2 lineage became widespread in the U.S. commercial swine population, subsequently spilling over into exhibition swine, and caused a majority of H3N2 variant (H3N2v) cases in humans in 2016 and 2017. A cluster of human H3N2v cases were reported at an agricultural fair in 2017 in Ohio, where 2010.1 H3N2 IAV was concurrently detected in exhibition swine. Genomic analysis showed that the swine and human isolates were nearly identical. In this study, we evaluated the propensity of a 2010.1 H3N2 IAV (A/swine/Ohio/A01354299/2017 [sw/OH/2017]) isolated from a pig in the agricultural fair outbreak to replicate in ferrets and transmit from swine to ferret. sw/OH/2017 displayed robust replication in the ferret respiratory tract, causing slight fever and moderate weight loss. Further, sw/OH/2017 was capable of efficient respiratory droplet transmission from infected pigs to contact ferrets. These findings establish a model for evaluating the propensity of swine IAV to transmit from pig to ferret as a measure of risk to the human population. The identification of higher-risk swine strains can then be targeted for control measures to limit the dissemination at human-swine interfaces to reduce the risk of zoonotic infections and to inform pandemic planning.

    IMPORTANCE A recently emerged lineage of human-like H3N2 (H3.2010.1) influenza A virus (IAV) from swine has been frequently detected in commercial and exhibition swine in recent years and has been associated with H3N2 variant cases in humans from 2016 and 2017. To demonstrate a model for characterizing the potential for zoonotic transmission associated with swine IAV, we performed an in vivo study of transmission between pigs infected with an H3.2010.1 H3N2 IAV and aerosol contact ferrets. The efficient interspecies transmission demonstrated for the H3.2010.1 IAV in swine emphasizes the need for further characterization of viruses circulating at the swine-human interface for transmission potential prior to human spillover and the development and implementation of more robust vaccines and control strategies to mitigate human exposure to higher-risk swine strains.

  • Articles of Significant Interest in This Issue [Spotlight]

  • Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35 [Genome Replication and Regulation of Viral Gene Expression]

  • Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain.

    IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

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    JVI Accepts: Articles Published Ahead of Print

  • Baculovirus transduction in mammalian cells is affected by the production of type I and III IFNs, which is mainly mediated by the cGAS-STING pathway [Gene Delivery]

  • The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of human epithelial cells HEK293 and HEK293 T, deficient in cGAS or in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted more relevant in the production of interferon (IFN)-bbeta; and IFN-1 in response to baculovirus infection. In human epithelial cells, IFN-1 production was also induced in a cGAS-independent and DNA-PK-dependent manner. Finally, we demonstrated that these cellular responses towards baculovirus infection affect the efficiency of transduction of baculovirus vectors.

    IMPORTANCE Baculoviruses are non-pathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

  • Identification of a functional small non-coding RNA encoded by African swine fever virus [Virus-Cell Interactions]

  • African swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs, against which there is no vaccine available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components including small non-coding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. In order to investigate the interplay between ASFV and sncRNAs, a study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to up to a 1 log reduction in ASFV growth indicating that ASFV utilises a virally-encoded small RNA to disrupt its own replication.

    IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic in Africa and Eastern Europe and rapidly emerging into Asia where it has led to the deaths of over a million pigs in the last 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterising the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterised a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

  • Porcine Reproductive and Respiratory Syndrome Virus promotes SLA-DR-mediated antigen presentation of non-structure proteins to evoke a non-neutralizing antibody response in vivo [Pathogenesis and Immunity]

  • The humoral immune response against PRRSV infection is characterized by a rapid induction of non-neutralizing antibodies (NAbs) against non-structure proteins (NSPs). Here, we systematically investigated the potential mechanism for the induction of PRRSV NSP-specific non-NAbs. Our data suggested that PRRSV-NSP-specific NAbs appeared within 10 days after PRRSV infection in vivo. In the in vitro model, functional upregulation of the SLA-DR was observed in bone marrow-derived dendritic cells (BM-DCs) and porcine alveolar macrophages (PAMs), whereas remarkable inhibition at the mRNA level was observed after infection by both PRRSV-1 and PRRSV-2 isolates. Notably, the inconsistency in SLA-DR expression between the mRNA and protein levels was resulted from the de-ubiquitination of SLA-DR via the OTU domain of PRRSV-NSP2 to inhibit ubiquitin-mediated degradation. Moreover, mass spectrometry-based immunopeptidome analysis identified immunopeptides originating from multiple PRRSV-NSPs within SLA-DR of PRRSV-infected BM-DCs. Meanwhile, these PRRSV-NSP-derived immunopeptides could be specifically recognized by serum from PRRSV-infected piglets. Notably, certain NSP-derived immunopeptides characterized in vitro could be identified from PAMs or hilar lymph nodes from PRRSV-infected piglets. More importantly, in vitro neutralizing assay indicated that serum antibodies against NSP-immunopeptides were unbale to neutralize PRRSV in vitro. Conversely, certain Structure Proteins (SPs)-derived immunopeptides were identified and could be recognize by pig hyperimmune serum against PRRSV, further indicates that NSP-derived antibodies response is non-protective in vivo. In conclusion, our data suggested that PRRSV infection interferes with the MHC-II molecule-mediated antigen presentation in APCs via promoting SLA-DR expression to present immunopeptides from PRRSV-NSPs, which contributes to the induction of non-NAbs antibodies in vivo.

    IMPORTANCE PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV Modified live vaccines (MLV) against heterogeneous PRRSV isolate, rapid induction of non-neutralizing antibodies (non-NAbs) against PRRSV-NSPs after MLV immunization or wild strains infection was one of the reasons hampered development of an effective vaccine. By using in vitro generated BM-DCs as models to understand the antigen presentation process of PRRSV and, our data indicated that PRRSV infection of BM-DCs promotes functional SLA-DR upregulation to present PRRRSV-NSPs- derived immunopeptides for evoking non-NAbs response in vivo. Our work not only uncovered novel mechanism regarding interference of host antigen presentation by PRRSV but also revealed novel insight for understanding the rapid production of non-neutralizing antibodies against PRRSV-NSPs, which may benefit for developing an effective PRRSV vaccine against PRRSV in future.

  • GBP5 is an interferon-induced inhibitor of respiratory syncytial virus [Virus-Cell Interactions]

  • Guanylate binding protein 5 (GBP5) belongs to the guanosine triphosphatase (GTPase) subfamily, which is mainly induced by interferon (IFN-) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. Here, we identified GBP5 as an effector of the anti-RSV activity of IFN- and found that in children, the weaker immune response, especially the weaker IFN- response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants at the C-terminus or lacking the C-terminus region, which impair GBP5 localisation in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localisation but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48-ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of the RSV.

    IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection is necessary to develop an effective strategy to control RSV infection. In this study, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.

  • Interferon inducible GTPase 1 impedes the dimerization of rabies virus phosphoprotein and restricts viral replication [Pathogenesis and Immunity]

  • Rabies, caused by rabies virus (RABV), is an ancient zoonosis and still a major public health problem for humans, especially in developing countries. RABV can be recognized by specific innate recognition receptors, resulting in the production of hundreds of interferon-stimulated genes (ISGs), which can inhibit viral replication at different stages. Interferon inducible GTPase 1 (IIGP1) is a mouse specific ISG and belongs to the immunity-related GTPases (IRGs) family. IIGP is reported to constrain intracellular parasite infection by disrupting the parasitophorous vacuole membrane. However, the role of IIGP1 in restricting viral replication has not been reported. In this present study, we found that IIGP1 was up-regulated in cells and in mouse brains upon RABV infection. Overexpression of IIGP1 limited RABV replication in cell lines and reduced viral pathogenicity in a mouse model. Consistently, deficiency of IIGP1 enhanced RABV replication in different parts of mouse brains. Furthermore, we found that IIGP1 could interact with RABV phosphoprotein (P protein). Mutation and immunoprecipitation analyses revealed that the Y128 site of P protein is critical for its interaction with IIGP1. Further study demonstrated that this interaction impeded the dimerization of P protein and thus suppressed RABV replication. Collectively, our findings for the first reveal a novel role of IIGP1 in restricting a typical neurotropic virus, RABV, which will provide a fresh insight into the function of this mouse specific ISG.

    IMPORTANCE Interferon and its downstream products, interferon-stimulated genes (ISG), are essential in defending against pathogen invasion. One of the ISGs, interferon inducible GTPase 1 (IIGP1), has been found to constrain intracellular parasite infection by disrupting their vacuole membranes. However, the role of IIGP1 in limiting viral infection is unclear. In this study, we show that infection with a typical neurotropic virus, rabies virus (RABV), can induce up-regulation of IIGP1, which in turn suppresses RABV by interacting with its phosphoprotein (P protein) and thus blocking the dimerization of P protein. Our study provides the first evidence that IIGP1 functions in limiting viral infection and provides a basis for comprehensive understanding of this important ISG.

  • A mechanism-based targeted screen to identify Epstein-Barr virus-directed antiviral agents [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal-cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that, through the activities of the viral protein kinase that is conserved across herpesviruses and two cellular proteins ATM and KAP1, a lytic cycle amplification loop is established nndash; and that disruption of this loop disables the EBV lytic cascade. We therefore devised a high throughput screening assay, screened a small molecule compound library, and identified seventeen candidates that impair the release of lytically-replicated EBV. The identified compounds will i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses and ii) be developed into anti-cancer agents as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for anti-herpesviral and anti-cancer drugs.

    IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even anti-herpesviral potential as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both anti-herpesviral and anti-cancer drugs.

  • Mosquito defensins enhance Japanese encephalitis virus infection by facilitating virus adsorption and entry within mosquito [Virus-Cell Interactions]

  • Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.

    IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection: mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.

  • The interaction interface of Mason-Pfizer monkey virus matrix and envelope proteins. [Structure and Assembly]

  • Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for HIV, the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane.

    To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used in the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pull-down assay. Through the combination of NMR spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C-terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N-terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.

    IMPORTANCE By a combination of NMR and mass spectroscopy of cross-linked peptides, we show that in contrast to HIV-1, the C-terminal residues of the unstructured cytoplasmic tail of M-PMV Env interact with MA. Based on biochemical data and molecular modeling, we propose that individual CT monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that membrane-bound MA domain of Gag recruits Env through interaction with full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively

  • Next-generation porcine intestinal organoids: an apical-out organoid model for swine enteric virus infection and immune response investigations [Virus-Cell Interactions]

  • The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both RTnndash;qPCR and IFA analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of IFN-aalpha;, IFN-1, ISG15, ISG58, TNF-aalpha;, and IL-6 in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines.

    IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The 3D porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I, and type III IFN antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.

  • Human Immunodeficiency Virus Type-1 and Methamphetamine Mediated Mitochondrial Damage and Neuronal Degeneration in Human Neurons [Virus-Cell Interactions]

  • Methamphetamine, a potent psychostimulant, is a highly addictive drug commonly used by persons living with HIV (PLWH), and its use can result in cognitive impairment and memory deficits long after its use is discontinued. Although the mechanism(s) involved with persistent neurological deficits are not fully known, mitochondrial dysfunction is a key component in methamphetamine neuropathology. Specific mitochondrial autophagy (mitophagy), and mitochondrial fusion and fission are protective quality control mechanisms that can be dysregulated in HIV infection and the use of methamphetamine can further negatively affect these protective cellular mechanisms. Here, we observed that treatment of human primary neurons (HPNs) with methamphetamine and HIV gp120 and Tat increase dynamin-related protein (DRP1)-dependent mitochondrial fragmentation and neuronal degeneration. Methamphetamine and HIV proteins increased microtubule-associated protein 1 light chain 3 beta-II (LC3B-II) lipidation and induced sequestosome 1 (SQSTM1, P62) translocation to damaged mitochondria. Additionally, the combination inhibited autophagic flux, increased reactive oxygen species (ROS) production and mitochondrial damage, and reduced microtubule associated protein-2 (MAP2) dendrites in human neurons. N-acetylcysteine (NAC), a strong antioxidant and ROS scavenger, abrogated DRP1-dependent mitochondrial fragmentation and neurite degeneration. Thus, we show that methamphetamine combined with HIV proteins inhibits mitophagy and induces neuronal damage, and NAC reverses these deleterious effects on mitochondrial function.

    IMPORTANCE Human and animal studies show that HIV infection combined with the long-term use of psychostimulants increase neuronal stress and the occurrence of HIV-associated neurocognitive disorders (HAND). On the cellular level, mitochondrial function is critical for neuronal health. In this study, we show that in human primary neurons, the combination of HIV proteins and methamphetamine increase oxidative stress, DRP-1 mediated mitochondrial fragmentation and neuronal injury manifested by a reduction in neuronal network and connectivity. The use of NAC, a potent antioxidant, reversed the neurotoxic effects of HIV and methamphetamine suggesting a novel approach to ameliorate the effects of HIV and methamphetamine associated cognitive deficits.

  • Specific Akt family members impair stress mediated transactivation of viral promoters and enhance neuronal differentiation: important functions for maintaining latency [Virus-Cell Interactions]

  • Neurotropic aalpha;-herpesvirinae subfamily members, bovine herpesvirus 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) for example, establish and maintain life-long latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part, because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency compared to reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, HSV-1 infected cell protein 0 (ICP0) promoter, and mouse mammary tumor virus long terminal repeat. Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.

    IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of alpha-herpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal Akt1 and Ak2, but not Akt3, impaired GR and dexamethasone mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2 stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of life-long latency.

  • Non-Nucleoside Inhibitors of Zika virus RNA-dependent RNA polymerase [Vaccines and Antiviral Agents]

  • Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects such as microcephaly in newborns and neurological disease like Guillain-Barreeacute; syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here we report the development of small non-nucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket ("N-pocket") located next to a putative hinge region between the thumb and the palm subdomains, that was originally described for dengue virus (DENV) RdRp. We first tested DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules and assessed their potency using both enzymatic and cell-based assays. The most potent compound has an IC50 value of 7.3 mmu;M and inhibits ZIKV replication in a cell-based assay with an EC50 value of 24.3 mmu;M. Importantly we report four high-resolution crystal structures detailing how these NNIs insert into the N-pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment and hydration of the N-pocket from ZIKV RdRp compared to DENV RdRp, that are crucial for the design of improved antiviral inhibitors against ZIKV.

    IMPORTANCE Zika virus belongs to the flavivirus family that comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The NS5 polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthetized inhibitors targeting the Zika virus NS5. We showed that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrate specific binding to an allosteric site within the polymerase called the N-pocket. This work paves the way for future structure-based design of potent compounds specifically targeting the ZIKV RNA polymerase activity.

  • L226Q mutation on influenza H7N9 virus hemagglutinin increases receptor-binding avidity and leads to biased antigenicity evaluation [Vaccines and Antiviral Agents]

  • Since the first outbreak in 2013, the influenza A (H7N9) virus has continued emerging and caused over five epidemic waves. Suspected antigenic changes of the H7N9 virus based on hemagglutination inhibition (HI) assay during the fifth outbreak have prompted the update of H7N9 candidate vaccine viruses (CVVs). In this study, we comprehensively compared the serological cross-reactivity induced by the hemagglutinins (HAs) of the earlier CVV A/Anhui/1/2013 (H7/AH13) and the updated A/Guangdong/17SF003/2016 (H7/GD16). We found that although H7/GD16 showed poor HI cross-reactivity to immune sera from mice and rhesus macaques vaccinated with either H7/AH13 or H7/GD16, the cross-reactive neutralizing antibodies between H7/AH13 and H7/GD16 were comparably high. Passive transfer of H7/AH13 immune sera also provided complete protection against the lethal challenge of H7N9/GD16 virus in mice. Analysis of amino acid mutations in the HAs between H7/AH13 and H7/GD16 revealed that L226Q substitution increases the HA binding avidity to sialic acid receptors on red blood cells, leading to decreased HI titers against viruses containing HA Q226 and thus resulted in a biased antigenic evaluation based on HI assay. These results suggest that amino acids located in the receptor-binding site could mislead the evaluation of antigenic variation by solely impact on the receptor-binding avidity to red blood cells without genuine contribution to antigenic drift. Our study highlights that viral receptor-binding avidity and combination of multiple serological assays should be taken into consideration in evaluating and selecting a candidate vaccine virus of H7N9 and other subtypes of influenza viruses.

    IMPORTANCE The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using HI assay. Other serological assays, such as MN assay, should be considered as a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.

  • Two genetic differences between closely-related Zika virus strains determine pathogenic outcome in mice [Pathogenesis and Immunity]

  • Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here we identify two strain-specific determinants of ZIKV virulence in mice. We found that H/PF/2013 caused 100% lethality in Ifnar1-/- mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1-/- Ifngr1-/- DKO mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G to T change at nucleotide 1965 producing a Val to Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both strains, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain, but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 non-coding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1-/- mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1-/- and Ifnar1-/- Ifngr1-/- DKO mice).

    IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here we identify two strain-specific determinants of ZIKV virulence which are evident only in Ifnar1-/- mice, not Ifnar1-/- Ifngr1-/- DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain, but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence, not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used strains of ZIKV, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.

  • SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles [Virus-Cell Interactions]

  • SERINC5 is a 10-12 transmembrane domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3-loop, a sCD4-induced epitope, and an N-linked glycan. By contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes.

    IMPORTANCE SERINC5 is a host-cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins, and this action is correlated with SERINC5's ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

  • Unpaired Guanosines in the 5' Untranslated Region of HIV-1 RNA Act Synergistically to Mediate Genome Packaging [Genome Replication and Regulation of Viral Gene Expression]

  • The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded, exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalence in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites using a chemical probe reverse-footprinting assay and as the major Gag binding sites using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in less than two-fold defects in packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA packaging, suggesting redundancy in Gag:RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 genome encapsidation.

    IMPORTANCE HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag:RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA.

  • Neuronal ablation of IFN{alpha}/{beta} signaling exacerbates CNS viral dissemination and impairs IFN{gamma} responsiveness in microglia/macrophages [Cellular Response to Infection]

  • IFNaalpha;/bbeta; signaling through the IFNaalpha;/bbeta; receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression pattern of factors associated with the IFNaalpha;/bbeta; pathway in different CNS resident cell populations implicate complex cooperative pathways in IFNaalpha;/bbeta; induction and responsiveness. Herein we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIaalpha;) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (MHV-A59) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice not only affected neurons in the forebrain, but also the mid-hind brain and spinal cords, but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs, but a decrease in mRNA levels of IFNaalpha;/bbeta; pathway genes as well as Il6, Tnf, and Il1bbeta; between days 4 and 6 post infection (p.i.). T cell accumulation and IFN production, an essential component of virus control, were not altered. However, IFN responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFNaalpha;/bbeta; signaling in neurons can worsen disease course and disrupt complex interactions between the IFNaalpha;/bbeta; and IFN pathways in achieving optimal antiviral responses.

    IMPORTANCE IFNaalpha;/bbeta; induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity and microglia/macrophage activation. However, the extent to which glial or neuronal signaling contributes to these diverse IFNaalpha;/bbeta; functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model this report demonstrates an essential role of IFNaalpha;/bbeta; receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN signaling, and prevent acute mortality. The results support that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFNaalpha;/bbeta; pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFNaalpha;/bbeta; and IFN signaling pathways to optimize antiviral IFN activity.

  • Optimized pseudotyping conditions for the SARS COV-2 Spike glycoprotein [Structure and Assembly]

  • The SARS COV-2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS COV-2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals.

    IMPORTANCE When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS COV-2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.

  • Drug-resistance mutation frequency of SGA-derived HIV-1 polymerase genomes in the CSF and plasma of HIV-1-infected individuals under non-suppressive therapy [Genetic Diversity and Evolution]

  • HIV-1 evolution in the CSF and plasma may result in discordant drug-resistance mutations (DRMs) in the compartments. Single genome amplification (SGA) was used to generate partial HIV-1 polymerase genomes in paired CSF and plasma from 12 HIV-1 positive CHARTER participants classified as neurocognitively unimpaired or with various degrees of HIV-Associated Neurocognitive Disorders (HAND). Subjects were viremic on combination antiretroviral therapy (cART). HIV-1 DRMs and phylogenetic characteristics were determined using the Stanford HIVdb program and phylogenetic analyses. Individual DRMs were identified more frequently in plasma than in paired CSF (p=0.0078). Significant differences in the ratios of DRMs in CSF and plasma were found in 3 individuals with HAND (3/7=43%). 2 HAND subjects (2/7=29%) demonstrated one DRM in CSF not identified in paired plasma. Longitudinal analyses (n=4) revealed significant temporal differences in the ratios of DRMs in the compartments. Statistically significant differences in the frequency of DRMs in the CSF and plasma are readily found in those on non-suppressive cART. While compartment-based DRM discordance was largely consistent with increased drug-selective pressures in the plasma, overrepresentation of DRMs in the CNS can occur. Underlying mechanisms of HAND are complex and multifactorial. The clinical impact of DRM discordance on viral persistence and HAND pathogenesis remains unclear and warrants further investigation in larger, longitudinal cohorts.

    Importance: Several antiretroviral agents do not efficiently enter the CNS and independent evolution of HIV-1 viral variants in the CNS and plasma can occur. We use single genome amplification (SGA) in cross-sectional and longitudinal analyses to uniquely define both the identity and relative proportions of drug resistance mutations (DRMs) on individual HIV-1 pol genomes in the cerebrospinal fluid (CSF) and plasma in individuals with incomplete viral suppression and known neurocognitive status. Statistically significant differences in the ratio of DRMs in the CSF and plasma were readily found in those on non-suppressive cART and overrepresentation of DRMs in the CNS can occur. Although questions about the clinical significance of DRM discordance remain, in the quest for viral eradication, it is important to recognize that a significant, dynamic, compartment-based DRM ratio imbalance can exist, as it has the potential to go unnoticed in the setting of standard clinical drug resistance testing.

  • hnRNP A1 and hnRNP A2 inhibit splicing to HPV16 splice site SA409 through a UAG-containing sequence in the E7 coding region [Genome Replication and Regulation of Viral Gene Expression]

  • Human papillomavirus type 16 (HPV16) 5'-splice site SD226 and 3'-splice site SA409 are required for production of the HPV16 E7 mRNAs, whereas unspliced mRNAs produce E6 mRNAs. The E6 and E7 proteins are essential in the HPV16 replication cycle but are also the major HPV16 proteins required for induction and maintenance of malignancy caused by HPV16 infection. Thus, a balanced expression of unspliced and spliced mRNAs are required for production of sufficient quantities of E6 and E7 proteins under physiological and pathophysiological conditions. If splicing becomes too efficient, the levels of unspliced E6 mRNAs will decrease below a threshold level that is no longer able to produce E6 protein quantities high enough to significantly reduce p53 protein levels. Similarly, if splicing becomes too inefficient, the levels of spliced E7 mRNAs will decrease below a threshold level that is no longer able to produce E7 protein quantities high enough to significantly reduce pRb protein levels. To determine how splicing between SD226 and SA409 is regulated, we have investigated how SA409 is controlled by the cellular proteins hnRNP A1 and hnRNP A2 - two proteins that have been shown previously to control HPV16 gene expression. We found that hnRNP A1 and A2 interacted directly and specifically with a C-less, 11-nucleotide RNA element located between HPV16 nucleotide positions 594 and 604 downstream of SA409. Overexpression of hnRNP A1 inhibited SA409 and promoted production of unspliced E6 mRNAs at the expense of the E7 mRNAs, whereas overexpression of hnRNP A2 inhibited SA409 to redirect splicing to SA742, a down-stream 3'-splice site that is used for generation of HPV16 E6^E7, E1 and E4 mRNAs. Thus, high levels of either hnRNP A1 or hnRNP A2 inhibited production of the promitotic HPV16 E7 protein. We show that the hnRNP A1 and A2 proteins control the relative levels of the HPV16 unspliced and spliced HPV16 E6 and E7 mRNAs and function as inhibitors of HPV16 E7 expression.

    IMPORTANCE Human papillomavirus type 16 (HPV16) belongs to the "high-risk"-group of HPVs and is causing a variety of anogenital cancers and head and neck cancer. The two HPV16 oncoproteins E6 and E7 prevent apoptosis and promote mitosis and are essential for completion of the HPV16 life-cycle and for transformation of the infected cell and maintenance of malignancy. E6 and E7 are produced from two mRNAs that are generated in a mutually exclusive manner by alternative splicing. While E6 protein is made from the unspliced mRNA, E7 is made from the spliced version of the same pre-mRNA. Since sufficient quantities of both E6 and E7 are required for malignant transformation, this intricate arrangement of gene expression renders E6 and E7 expression vulnerable to external interference. Since antiviral drugs to HPV16 are not available, a detailed knowledge of the regulation of HPV16 E6 and E7 mRNA splicing may uncover novel targets for therapy.

  • Surprisingly Effective Priming of CD8+ T cells by Heat-Inactivated Vaccinia Virus Virions [Pathogenesis and Immunity]

  • Robust priming of CD8+ T cells by viruses is considered to require infection and de novo expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. Contrary to this dogma we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8+ T cell responses when the virus was rendered non-infectious by heat. More surprisingly, in some cases these responses were similar in magnitude to those primed by infectious virus administered at an equivalent dose. Next we tested whether this was a special property of particular antigens and their epitopes and found that foreign epitopes tagged onto three different VACV virion proteins were able to elicit CD8+ T cell responses irrespective of whether the virus was viable or heat-killed. Further, the polyfunctionality and cytotoxic ability of the CD8+ T cells primed by these VACVs was equivalent irrespective of whether they were administered to mice as inactivated or live viruses. Finally, we used these VACVs in prime-boost combinations of inactivated and live virus and found that priming with dead virus before a live booster was the most immunogenic regime. We conclude that VACV virions themselves can be efficient vectors for targeting antigens to dendritic cells for effective priming of CD8+ T cells, even when rendered non-infectious and speculate that this might also be the case for other viruses.

    Importance: The design of viral vectored vaccines is often considered to require a trade-off between efficacy and safety. This is especially the case for vaccines that aim to induce killer (CD8+) T cells, where there is a well-established dogma that links infection in vaccinated individuals with effective induction of immunity. However, we found that some proteins of vaccinia virus generate strong CD8+ T cell responses even when the virus preparation was inactivated by heat prior to administration as a vaccine. We took advantage of this finding by engineering a new vaccine vector virus that could be used as an inactivated vaccine. These results suggest that vaccinia virus may be a more versatile vaccine vector than previously appreciated and that in some instances safety can be prioritized by the complete elimination of viral replication without a proportional loss of immunogenicity.

  • Divergent symptoms caused by geminivirus-encoded C4 proteins correlate with their ability to bind NbSK{eta} [Cellular Response to Infection]

  • Geminiviruses induce severe developmental abnormalities in plants. The C4/AC4 protein encoded by geminiviruses, especially by those not associated with betasatellites, functions as a symptom determinant through hijacking a shaggy-related protein kinase (SK) and interfering with its functions. Here, we report that the symptom determinant capability of C4 proteins encoded by different geminiviruses is divergent and tightly correlated with their ability to interact with SK from Nicotiana benthamiana (NbSK). Swap of the mini-domain of tomato leaf curl Yunnan virus (TLCYnV) C4 critical for the interaction with NbSK increased the capacity of the C4 proteins encoded by tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) to induce symptoms. The severity of symptoms induced by recombinant TYLCCNV C4 or TbCSV C4 correlates with the amount of NbSK tethered to the plasma membrane by the viral protein. Moreover, a recombinant TYLCCNV harboring the mini-domain of TLCYnV C4 induces more severe symptoms than wild-type TYLCCNV. Thus, this study provides new insights into the mechanism by which different geminivirus-encoded C4 proteins possess divergent symptom determinant capabilities.

    IMPORTANCE Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases in many economically important crops world-wide. Geminivirus encoded C4 protein is a multifunctional protein. In this study, we found that the C4 proteins from different geminiviruses showing a differential ability to interact with NbSK, which correlated with their symptom determinant capability. Moreover, a mini-domain of tomato leaf curl Yunnan virus (TLCYnV) C4 indispensable for interacting with NbSK and tethering it to the plasma membrane to lead to symptom induction was determined. Supporting to these, a recombinant geminivirus carrying the mini-domain of TLCYnV C4 induced more severe symptoms than its original one. Therefore, these findings expand the scope of the interaction of NbSK and C4-mediated symptom induction and contribute to further understanding of the multiple roles of C4.

  • An optimized reverse genetics system suitable for efficient recovery of simian, human and murine-like rotaviruses [Genome Replication and Regulation of Viral Gene Expression]

  • An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 RVA segment-specific (+)ssRNAs, a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Secondly, a genetically modified MA104 cell line was used in which several compounds of the innate immune were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (gge;80% success rate) and could not be reliably rescued using several recently published RG strategies (llt;20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low replicating attenuated vaccine candidates or low cell culture passage clinical isolates from humans or animals.

    IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies and expression vectors.

  • The first nonmammalian pegivirus demonstrates efficient in vitro replication and high lymphotropism [Pathogenesis and Immunity]

  • Members of the Pegivirus genus, family Flaviridiae, widely infect humans and other mammals, including nonhuman primates, bats, horses, pigs, and rodents, but are not associated with disease. Here, we report a new, genetically distinct pegivirus in goose (Anser cygnoides), the first identified in a non-mammalian host species. Goose pegivirus (GPgV) can be propagated in goslings, embryonated goose eggs, and primary goose embryo fibroblasts and is thus the first pegivirus that can be efficiently cultured in vitro. Experimental infection of GPgV in goslings via intravenous injection revealed robust replication and shown high lymphotropism. Analysis of the tissue tropism of GPgV revealed that the spleen and thymus were the organs bearing the highest viral loads. Importantly, GPgV could promote clinical manifestations of goose parvovirus infection, reducing weight gain and 7% mortality. This finding contrasts with the lack of pathogenicity that is characteristic of previously reported pegiviruses.

    IMPORTANCE Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, but are described as causing persistent infection and lacking pathogenicity. The efficiency of in vitro replication system for pegivirus is poor limiting investigation into viral replication steps. Because of that, the pathogenesis, cellular tropism, route of transmission, biology, and epidemiology of pegiviruses remain largely uncovered. Here, we report a phylogenetically distinct goose pegivirus (GPgV) which should be classified as a new species. GPgV proliferated in cell culture in a species- and cell-type specific manner. Animal experiments shown GPgV lympho-tropism and promotes goose parvovirus clinical manifestations. This study provides the first cell culture model for pegivirus, opening new possibilities for studies of pegivirus molecular biology. More importantly, our finding stand in contrast to the lack of identified pathogenicity of previously reported pegiviruses, which sheds lights on the pathobiology of pegivirus.

  • A calcium sensor discovered in Bluetongue virus non-structural protein 2 is critical for virus replication [Virus-Cell Interactions]

  • Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways, however, to date no Ca2+ binding protein has been reported in Bluetongue virus (BTV), the causative agent of Bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV non-structural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by Alanine. Using Circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in presence of Ca2+, revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain which wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus and that the NS2 phosphorylation level and assembly of VIBs were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.

    IMPORTANCE After entering the host cells viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells members of Reoviridae family form an inclusion body like structure known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through non-structural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium binding EF hand like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

  • Partitiviruses infecting Drosophila melanogaster and Aedes aegypti exhibit efficient biparental vertical transmission [Genetic Diversity and Evolution]

  • Partitiviruses are segmented, multipartite dsRNA viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of Drosophila melanogaster. To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over three years, with between 63-100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion but vertical transmission from either infected females or infected males was ~100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors.

    IMPORTANCE Galbut virus is a recently discovered partitivirus that is extraordinarily common in wild populations of the model organism Drosophila melanogaster. Like most viruses discovered through metagenomics, most of the basic biological questions about this virus remain unanswered. We found that galbut virus, along with a closely related partitivirus found in Aedes aegypti mosquitoes, is transmitted from infected females or males to offspring with ~100% efficiency and can be maintained in laboratory colonies over years. This efficient transmission mechanism likely underlies the successful spread of these viruses through insect populations. We created Drosophila lines that contained galbut virus as the only virus infection and showed that these flies can be used as a source for experimental infections. This provides insight into how arthropod-infecting partitiviruses may be maintained in nature and sets the stage for exploration of their biology and potential utility.

  • Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) infections in 3-D human airway tissues expose an interesting dichotomy in viral replication, spread, and inhibition by neutralizing antibodies [Virus-Cell Interactions]

  • Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two of the leading causes of respiratory infections in children, elderly, and immunocompromised patients worldwide. There is no approved treatment for HMPV and only one prophylactic treatment against RSV; palivizumab, for high risk infants. Better understanding of the viral lifecycles in a more relevant model system may help identify novel therapeutic targets. By utilizing 3-D human airway tissues to examine viral infection in a physiologically-relevant model system, we showed that RSV infects and spreads more efficiently than HMPV, with the latter requiring higher MOIs to yield similar levels of infection. Apical ciliated cells were the target for both viruses, but RSV apical release was significantly more efficient than HMPV. In RSV- or HMPV-infected cells, cytosolic inclusion bodies containing the nucleoprotein, phosphoprotein, and respective viral genomic RNA were clearly observed in HAE culture. In HMPV-infected cells, actin-based filamentous extensions were more common (35.8%) than those found in RSV-infected cells (4.4%). Interestingly, neither RSV nor HMPV formed syncytia in HAE tissues. Palivizumab and nirsevimab effectively inhibited entry and spread of RSV in HAE tissues, with nirsevimab displaying significantly higher potency compared with palivizumab. In contrast, 54G10 completely inhibited HMPV entry but only modestly reduced viral spread, suggesting HMPV may use alternative mechanisms for spread. These results represent the first comparative analysis of infection by the two pneumoviruses in a physiologically-relevant model, demonstrating an interesting dichotomy in the mechanisms of infection, spread, and consequent inhibition of the viral lifecycles by neutralizing monoclonal antibodies.

    Importance

    Respiratory syncytial virus and human metapneumovirus are leading causes of respiratory illness worldwide, but limited treatment options are available. To better target these viruses, we examined key aspects of the viral lifecycle in 3-D human airway tissues. Both viruses establish efficient infection through the apical surface, but efficient spread and apical release was seen for RSV but not HMPV. Both viruses form inclusion bodies, minimally composed of N, P and vRNA, indicating that these structures are critical for replication in this more physiological model. HMPV formed significantly more long, filamentous actin-based extensions in HAE tissues compared with RSV, suggesting HMPV may promote cell-to-cell spread via these extensions. Lastly, RSV entry and spread were fully inhibited by neutralizing antibodies palivizumab and the novel nirsevimab. In contrast, while HMPV entry was fully inhibited by 54G10, a neutralizing antibody, spread was only modestly reduced, further supporting a cell-to-cell spread mechanism.

  • Localization of the interaction site of herpes simplex virus glycoprotein D (gD) on the membrane fusion regulator, gH/gL [Structure and Assembly]

  • A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL blocking MAbs were located within flexible regions of the gH N-terminus and the gL C-terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.

    IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with receptor, activates the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion which identifies targets for vaccine development.

  • T cell receptor diversity and lineage relationship between virus-specific CD8 T cell subsets during chronic LCMV infection [Cellular Response to Infection]

  • Recent studies on chronic viral infections have defined a novel PD-1+ TCF1+ stem-like CD8 T cell subset that gives rise to the terminally differentiated exhausted CD8 T cells. In this study, we performed T cell receptor (TCR)bbeta; sequencing of virus-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection to examine the TCR diversity and lineage relationship of these two functionally distinct subsets. We found that ggt;95% of the TCR repertoire of the exhausted CD8 T cell subset was shared with the stem-like CD8 T cells. The TCR repertoires of both CD8 T cell subsets were composed mostly of a few dominant clonotypes but there was slightly more breadth and diversity in the stem-like CD8 T cells compared to their exhausted counterpart (~40 versus ~15 GP33+ clonotypes; ~20 versus ~7 GP276+ clonotypes). Interestingly, the breadth of the TCR repertoire was broader during the early stages (day 8) of the chronic infection compared to the later stages (day 45-60) showing that there was a narrowing of the TCR repertoire during chronic infection (~2-fold GP33+ and GP276+ stem-like subset; ~10-fold GP33+ and ~5-fold GP276+ exhausted subset). In contrast, during acute LCMV infection the TCR repertoire was much broader in both GP33-specific effector (~160 clonotypes) and memory CD8 T cells (~160 clonotypes). Overall, our data demonstrate that the virus-specific CD8 T cell TCR repertoire is broad and remains stable after acute LCMV infection, but it contracts and is narrower during chronic infection. Our study also shows that the repertoire of the exhausted CD8 T cell subset is almost completely derived from the stem-like CD8 T cell subset during established chronic LCMV infection.

    IMPORTANCE CD8 TCR repertoires responding to chronic viral infections (HIV, HCV, EBV, and CMV) have limited breadth and diversity. How these repertoires change and are maintained throughout the chronic infection are unknown. We thus characterized the LCMV-specific CD8 TCR repertoires of stem-like and terminally exhausted subsets generated during chronic LCMV infections. During chronic LCMV infections, the repertoires started diverse but became more clonal at the late time point. Further, the exhausted subset was composed of dominant clonotypes that were shared with the stem-like subset. Together, we demonstrate that the TCR repertoire contracts over time and is almost exclusively derived from the stem-like subset late during the persistent viral infection. Our data suggest that dominant clonotypes in the exhausted subset are derived from a diverse pool of stem-like clonotypes which may be contributing to the clonality observed during chronic viral infections.

  • Uncovering the role of the E1 protein in different stages of HPV18 genome replication [Genome Replication and Regulation of Viral Gene Expression]

  • The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication nndash; initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2aalpha; expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins, and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function.

    IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes, maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases nndash; replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

  • Porcine reproductive and respiratory syndrome virus structural protein GP3 regulates claudin 4 to facilitate the early stages of infection [Virus-Cell Interactions]

  • Claudins (CLDN) are a family of proteins that represent the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Several types of viruses make full use of CLDN to facilitate its entry into cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry. In this study, we found that CLDN4 functions as an anti-PRRSV factor by blocking its absorption during the early stages of infection. The small extracellular loop (ECL2) of CLDN4 restricted the viral particles outside of cells by binding to GP3. A novel function of GP3-mediated regulation of CLDN4 transcription was suggested. CLDN4 can be decreased through downregulating the level of CLDN4 transcription by ubiquitinating the transcription factor, SP1. The mechanism by which HP-PRRSV infects the epithelium was proposed. Importantly, ECL2 was found to block PRRSV absorption, infection, and neutralize the virus. A more in-depth understanding of PRRSV infection is described and novel therapeutic antiviral strategies are discussed.

    IMPORTANCE

    In the present study, the role of CLDN4 in PRRSV infection was studied. The results showed that CLDN4 blocked the absorption into the cells and restricted extracellular viral particles via the interaction between the CLDN4 small extracellular loop, ECL2, and the viral surface protein, GP3. GP3 was found to downregulate CLDN4 through ubiquitinating the transcription factor, SP1, to facilitate viral entry. The mechanism by which HP-PRRSV infects the epithelium is suggested. A novel function of GP3 in regulating gene transcription was discovered. Moreover, ECL2 could block PRRSV absorption and infection, as well as neutralize the virus in the supernatant, which may lead to the development of novel therapeutic antiviral strategies.

  • The ASM Journals Committee Values the Contributions of Black Microbiologists [Editorial]

  • Black lives matter. The ongoing problem of police brutality and the resulting deaths of George Floyd (1), Breonna Taylor (2), and many other Black people in the United States (3) has rightly shaken the country. Acts of racism should cause us to question the level to which we have personally participated in the systems of racial inequity that facilitate such acts. We all have an obligation to identify the ways that systemic racism functions in our society and in science. As scientists, we prefer to believe that we are driven by data and are immune to such detrimental behaviors. Yet, if we are honest, we know that this is not always true.

  • The Molluscum Contagiosum Gene MC021L Partially Compensates for the Loss of Its Vaccinia Virus Homolog F13L. [Virus-Cell Interactions]

  • Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network, and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane is a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV, and plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope tagged version of MC021L. The resulting virus (vMC021L-HA) had a small plaque phenotype compared to vF13L-HA, but larger than vF13L. Localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced similar amounts of EV compared to vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding, and an increase in dissolution, both of which correlated with a small plaque phenotype.

    IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in-turn, is essential for subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV is produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L, termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate, and we show that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV, and further delineates the dual role of F13 during infection.

  • Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. siRNA-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in assembly step without affecting viral entry, HCV IRES-mediated translation, and replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs) and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating phosphorylation of cortactin at tyrosine residue 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.

    IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in virus life cycle has not yet been fully understood. The most significant finding is that cortactin strongly interacted with both HCV core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the LDs with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosine 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

  • Characterization of Self-processing Activities and Substrate Specificities of Porcine Torovirus 3C-like Protease [Structure and Assembly]

  • The 3C-like protease (3CLpro) of Nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV, family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically release itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites, and identified (FXXQA/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the anti-nidovirus drugs.

    IMPORTANCE

    Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a Glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the Phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

  • Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site [Cellular Response to Infection]

  • The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV) nndash; a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal-turbinate tissue viability and physiological functionality were preserved for at least seven days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal- and vascular endothelial cells within the tissue. Importantly, functional antiviral and pro-leukocyte-chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial-cell related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal-mucosal and placental tissues, representing the viral entry- and the maternal-to-fetal transmission site, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.

    IMPORTANCE HCMV is a ubiquitous human pathogen, causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here we describe the establishment and use of a novel nasal turbinate organ culture, to study the initial steps of viral infection and the consequent innate immune responses, within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially other viruses.

  • Contribution of segment 3 to the acquisition of virulence in contemporary H9N2 avian influenza viruses [Pathogenesis and Immunity]

  • H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example, H7N9. In recent years it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses.

    Importance

    Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this llsquo;fitness'. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions nndash; however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

  • Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein [Genome Replication and Regulation of Viral Gene Expression]

  • The human papillomavirus (HPV) E2 protein is a key regulator of viral transcription and replication. In this study, we demonstrate that the non-receptor tyrosine kinase Pyk2 phosphorylates tyrosine 131 in the E2 transactivation domain. Both depletion of Pyk2 and treatment with a Pyk2 kinase inhibitor increased viral DNA content in keratinocytes that maintain viral episomes. The tyrosine to glutamic acid (E) mutant Y131E which may mimic phosphotyrosine failed to stimulate transient DNA replication, and genomes with this mutation were unable to establish stable episomes in keratinocytes. Using co-immunoprecipitation assays, we demonstrate that the Y131E is defective for binding to the C-terminal motif (CTM) of Bromodomain-containing protein 4 (Brd4). These data imply HPV replication depends on E2 Y131 interaction with the pTEFb binding domain of Brd4.

    Importance

    Human papillomaviruses are the major causative agents of cervical, oral and anal cancers. The present study demonstrates that the Pyk2 tyrosine kinase phosphorylates E2 at tyrosine 131, interfering with genome replication. We provide evidence that phosphorylation of E2 prevents binding to the Brd4-CTM. Our findings add to the understanding of molecular pathways utilized by the virus during its vegetative lifecycle and offers insights into the host-virus interactome.

  • NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases [Genetic Diversity and Evolution]

  • Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3'-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.

    IMPORTANCE Intensive studies on HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatments escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens on the possibility of adapting to the optimisation of further functionalities exerted by the same protein. Such property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

  • In vitro quantification of the effects of IP6 and other small polyanions on immature HIV-1 particle assembly and core stability [Structure and Assembly]

  • Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexakisphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 lifecycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly co-factors or stabilizers of viral particles.

    IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexakisphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral lifecycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

  • Murine cytomegalovirus M25 proteins sequester the tumor suppressor protein p53 in nuclear accumulations [Virus-Cell Interactions]

  • To ensure productive infection herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the beta-herpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by co-immunoprecipitation. Moreover, the proteins co-localized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wildtype MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. Contrary to the higher abundance of the p53 protein in wildtype MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the M25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53.

    IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

  • ISGylation of hepatitis C virus NS5A protein promotes viral RNA replication via the recruitment of cyclophilin A [Genome Replication and Regulation of Viral Gene Expression]

  • Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called 'ISGylation'. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A-ISGylation in HCV replication is pro-viral or anti-viral. Here we investigated the role of NS5A-ISGylation in HCV replication by using HCV RNA replicons that have a mutation at each lysine (Lys) residue of NS5A protein. Immunoblot analyses revealed that five Lys residues (K44, K68, K166, K215, and K308) of 14 Lys residues within NS5A (1b, Con1) have the potential to accept ISGylation. We tested the NS5A-ISGylation among different HCV genotypes and observed that the NS5A of all of the HCV genotypes accept ISGylation at the multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that the residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A-ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence suggesting that NS5A-ISGylation derived from all of HCV genotypes enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A-ISGylation functions as a pro-viral factor and promotes HCV replication via the recruitment of CypA.

    IMPORTANCE

    Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type-I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible, ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here we demonstrate that HCV NS5A protein accepts ISG15-conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A-ISGylation. We obtained evidence suggesting that NS5A-ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.

  • Structure of Merkel cell polyomavirus capsid and interaction with its glycosaminoglycan attachment receptor [Structure and Assembly]

  • Merkel cell polyomavirus (MCPyV) is a human double-stranded DNA tumor virus. MCPyV cell entry is unique among the polyomavirus family as it requires the engagement of two types of glycans, sialylated oligosaccharides and sulfated glycosaminoglycans (GAGs). Here, we present crystallographic and cryo-electron microscopic structures of the icosahedral MCPyV capsid and analysis of its glycan interactions via NMR spectroscopy. While sialic acid binding is specific for aalpha;2-3-linked sialic acid and mediated by the exposed apical loops of the major capsid protein VP1, a broad range of GAG oligosaccharides bind to recessed regions between VP1 capsomers. Individual VP1 capsomers are tethered to one another by an extensive disulfide network that differs in architecture from previously-described interactions for other PyVs. An unusual C-terminal extension in MCPyV VP1 projects from the recessed capsid regions. Mutagenesis experiments show that this extension is dispensable for receptor interaction.

    IMPORTANCE

    The MCPyV genome was found to be clonally integrated in 80% of Merkel cell carcinoma (MCC), a rare but aggressive form of human skin cancer, strongly suggesting that this virus is tumorigenic. In the metastasizing state the course of disease is often fatal, especially in immunocompromised individuals, as reflected by the high mortality rate of 33-46% and low 5-year survival rate (llt; 45%). The high seroprevalence of about 60% makes MCPyV a serious healthcare burden and illustrates the need for targeted treatments. In this study we present the first high-resolution structural data of this human tumor virus and demonstrate that the full capsid is required for the essential interaction with its GAG receptor(s). Together this data can be used as a basis for future strategies in drug development.

  • Role of escape mutant-specific T cells in suppression of HIV-1 replication and co-evolution with HIV-1 [Pathogenesis and Immunity]

  • The accumulation of HIV-1 escape mutations affects HIV-1 control by HIV-1-specific T cells. Some of these mutations can elicit escape mutant-specific T-cells, but it still remains unclear whether they can suppress the replication of HIV-1 mutant viruses. It is known that HLA-B*52:01-restricted RI8 (Gag 275-282, RMYSPTSI) is a protective T cell epitope in HIV-1 subtype B-infected Japanese individuals, though 3 Gag280A/S/V mutations are found in 26% of them. Gag280S and Gag280A were HLA-B*52:01-associated mutations; whereas Gag280V was not, implying a different mechanism for the accumulation of Gag280 mutations. We here investigated the co-evolution of HIV-1 with RI8-specific T-cells and suppression of HIV-1 replication by its escape mutant-specific T-cells both in vitro and in vivo. The HLA-B*52:01+ individuals infected with Gag280A/S mutant viruses failed to elicit these mutant epitope-specific T-cells, whereas those with the Gag280V mutant one effectively elicited RI8-6V mutant-specific T-cells. These RI8-6V-specific T cells suppressed the replication of Gag280V virus and selected wild-type virus, suggesting a mechanism affording no accumulation of the Gag280V mutation in the HLA-B*52:01+ individuals. The responders to wild-type (RI8-6T) and RI8-6V mutant peptides had significantly higher CD4 counts than non-responders, indicating that the existence of not only RI8-6T-specific T cells but also RI8-6V-specific ones was associated with a good clinical outcome. The present study clarified the role of escape mutant-specific T cells in HIV-1 evolution and in the control of HIV-1.

    Importance Escape mutant-specific CD8+ T-cells were elicited in some individuals infected with escape mutants, but it is still unknown that these CD8+ T-cells can suppress HIV-1 replication. We clarified that Gag280V mutation were selected by HLA-B*52:01-restricted CD8+ T-cells specific for GagRI8 protective epitope whereas the Gag280V virus could frequently elicit GagRI8-6V mutant-specific CD8+ T-cells. GagRI8-6V mutant-specific T-cells had a strong ability to suppress the replication of the Gag280V mutant virus both in vitro and in vivo. In addition, these T cells contributed to the selection of wild-type virus in HLA-B*52:01+ Japanese individuals. We for the first time demonstrated that escape mutant-specific CD8+ T-cells can suppress HIV-1 replication and play an important role in the coevolution with HIV-1. Thus, the present study highlighted an important role of escape mutant-specific T-cells in the control of HIV-1 and co-evolution with HIV-1.

  • Analysis of virus population profiles within pigs infected with virulent Classical Swine Fever viruses; evidence for bottlenecks in transmission but absence of tissue specific virus variants [Genetic Diversity and Evolution]

  • Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that varies between strains of different virulence. In the highly virulent CSFV Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV Koslov (here called vKos_SL), to generate modified CSFVs with substitutions at residues 763/764 (vKos_LL, vKos_SP and vKos_LP). The properties of these mutant viruses, in comparison to vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF but the vKos_LP produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue dependent quasispecies differentiation occurred within inoculated pigs, however, clear evidence for transmission bottle-necks to contact animals was observed, with subsequent loss of sequence diversity.

    IMPORTANCE The surface exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

  • Critical residues and contacts within domain IV of Autographa californica multiple nucleopolyhedrovirus GP64 contribute to its refolding during membrane fusion [Structure and Assembly]

  • Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In postfusion structure, GP64 is composed of five domains (domain I-V). Domain IV (374-407 aa) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to substitute each of the residues and the contacts within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, substitution of a single amino acid has no significant impact on GP64. However, substitution of R392 or disrupting the contact N381-N385, N384-Y388, N385-W393, or K389-W393 resulted in poor cell surface expression and fusion loss of the modified GP64, whereas substitution of E390 or G391, or disrupting the contact N381-K389, N381-Q401, or N381-I403 reduced the cell surface level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, substitution of N407 or disrupting the contact D404-S406 appears to restrict fusion pore expansion without affecting expression. Combined with the identification of these constructs remaining stable prefusion conformation or dramatically less efficient transition from a prefusion to postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion.

    Importance Baculoviruses GP64 is grouped with rhabdoviruses G, herpesviruses gB, and thogotoviruses glycoproteins as class III viral fusion proteins. In their postfusion structures, these proteins contain five domains (domain I-V). Distinguished from domain IV of rhabdoviruses G and herpesviruses gB that composed of bbeta;-sheets, domain IV of GP64 is a loop region and the same domain in thogotovirues glycorproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that the highly conserved residues and the contacts within domain IV of AcMNPV GP64 are necessary for low-pH triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.

  • NS5 Sumoylation Directs Nuclear Responses that Permit Zika Virus to Persistently Infect Human Brain Microvascular Endothelial Cells [Virus-Cell Interactions]

  • Zika virus (ZIKV) is cytopathic to neurons and persistently infects brain microvascular endothelial cells (hBMECs), that normally restrict viral access to neurons. Despite replicating in the cytoplasm, ZIKV and Dengue virus (DENV) polymerases, NS5 proteins, are predominantly trafficked to the nucleus. We found that a SUMO interaction motif in ZIKV and DENV NS5 proteins directs nuclear localization. Dissimilarly, ZIKV NS5 formed discrete punctate nuclear bodies (NBs), while DENV NS5 was uniformly dispersed in the nucleoplasm. However, mutating one DENV NS5 SUMO site (K546R) localized the NS5 mutant to discrete NBs, and NBs formed by the ZIKV NS5 SUMO mutant (K252R) were restructured and in discrete protein complexes. In hBMECs, NBs formed by STAT2 and promyelocytic leukemia (PML) protein are present constitutively and enhance innate immunity. During infection or NS5 expression we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of IFN stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 to regulate hBMEC cell cycle transcriptional responses. Our data reveals NS5 SUMO motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs.

    Importance

    ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here we demonstrate that flavivirus specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization, and that SUMO sites regulate NS5 NB complex constituents, assembly and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs and direct PML degradation. ZIKV NS5 SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons.

  • A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes [Virus-Cell Interactions]

  • The unwinding of double-stranded RNA intermediates is critical for replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that Motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in Motif V exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of Motif V in viral replication using T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo. We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection, and increased cytopathogenicity in cell culture as compared to WT Kunjin. Similarly, decreased infection rates in surviving S411A-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these data indicate that NS3 Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.

    IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years demonstrating they remain a global threat. Currently, there are no approved anti-viral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigate the role of the Kunjin virus helicase on infection in cell culture and in vivo. This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

  • Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures [Cellular Response to Infection]

  • The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNFaalpha;, CXCL8, and identified NF-B and ATF-4 as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pre-treatment and post-treatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.

    IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways, that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cultures induce a strong pro-inflammatory cytokine response yet block the production of type I and III IFNs. to SARS-CoV-2. However, treatment of airway cultures with the immune molecules, type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

  • Antiviral Properties of the LSD1 Inhibitor SP-2509 [Genome Replication and Regulation of Viral Gene Expression]

  • The LSD1 demethylase targets cellular proteins including histone H3, p53, E2F, and Dnmt1 and is involved in the regulation of gene expression, DNA replication, the cell cycle, and the DNA damage response. LSD1 catalyzes demethylation of histone H3K9 associated with HSV-1 immediate early (IE) promoters and is necessary for IE gene expression, viral DNA replication, and reactivation from latency. We previously found that LSD1 associates with HSV-1 replication forks and replicating viral DNA, suggesting that it may play a direct role in viral replication or coupled processes. We investigated the effects of the LSD1 inhibitor SP-2509 on the HSV-1 life cycle. Unlike previously investigated LSD1 inhibitors TCP and OG-L002, which covalently attach to the LSD1 cofactor FAD to inhibit demethylase activity, SP-2509 has previously been shown to inhibit LSD1 protein-protein interactions. We found that SP-2509 does not inhibit HSV-1 IE gene expression or transcription factor and RNA polymerase II (Pol II) association with viral DNA prior to the onset of replication. However, SP-2509 does inhibit viral DNA replication, late gene expression, and virus production. We used EdC-labeling of nascent viral DNA to image aberrant viral replication compartments that form in the presence of SP-2509. Treatment resulted in the formation of small replication foci that colocalize with replication proteins but are defective for Pol II recruitment. Taken together, these data highlight a potential new role for LSD1 in the regulation of HSV-1 DNA replication and gene expression after the onset of DNA replication.

    IMPORTANCE Treatment of HSV-1 infected cells with SP-2509 blocked viral DNA replication, gene expression after the onset of DNA replication, and virus production. These data support a potential new role for LSD1 in the regulation of viral DNA replication and successive steps in the virus life cycle and further highlight the promising potential to utilize LSD1 inhibition as an antiviral approach.

  • Evolutionary arms race between virus and host drives genetic diversity in bat SARS related coronavirus spike genes [Genetic Diversity and Evolution]

  • The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we have identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiency in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS- and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that some key residues were under positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics.

    Importance Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoV in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS- and SARSr-CoV infection but with different binding affinity to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity of spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests a long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.

  • Herpes Simplex Virus Organizes Cytoplasmic Membranes to Form a Viral Assembly Center in Neuronal Cells [Structure and Assembly]

  • Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms multiple, dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is a major site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization.

    Importance Herpesvirus particles are complex and contain many different proteins that must come together in an organized and coordinated fashion. Many viruses solve this coordination problem by creating a specialized assembly factory in the host cell, and the formation of such factories provides a promising target for interfering with virus production. Herpes simplex virus 1 (HSV-1) infects several types of cells, including neurons, but has not previously been shown to form such an organized factory in the non-neuronal cells in which its assembly has been best studied. Here we show that HSV-1 forms an organized assembly factory in neuronal cells, and we identify some of the viral and host cell factors that are important for its formation.

  • N-linked glycan sites on the influenza NA head domain are required for efficient IAV incorporation and replication [Structure and Assembly]

  • N-linked glycans commonly contribute to secretory protein folding, sorting and signaling. For enveloped viruses such as the influenza A virus (IAV), the addition of large N-linked glycans can also prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.

    IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 vs H3N2) and species of origin, with human derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.

  • Sequential phosphorylation of the HCV NS5A protein depends on NS3-mediated auto-cleavage between NS3 and NS4A [Genome Replication and Regulation of Viral Gene Expression]

  • Replication of the genotype 2 hepatitis C virus (HCV) requires hyper-phosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyper-phosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyper-phosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in the above sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wildtype NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction, too, abolished NS5A hyper-phosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant that resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyper-phosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated auto-cleavage at the NS3-4A junction is critical to NS5A hyper-phosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyper-phosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.

    Importance For twenty some years, the HCV protease NS3 was implicated in NS5A hyper-phosphorylation. We now show that it was the NS3-mediated cis-cleavage at the NS3-4A junction that permits NS5A hyper-phosphorylation at serine 2201, 2208, 2211, and 2214, leading to hyper-phosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at the above serine residues right after NS3-4A cleavage and before NS5A was released from the NS4A-5A polyprotein. Our data suggest that the dual functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyper-phosphorylation.

  • Anti-chikungunya virus monoclonal antibody inhibiting viral fusion and release [Vaccines and Antiviral Agents]

  • Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae. Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a bbeta;-ribbon connector, and that its bound Fab on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45 mmu;m filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.

    IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for chikungunya virus (CHIKV) infection is available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane-fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

  • Immune escape adaptive mutations in the H7N9 avian influenza hemagglutinin protein increase virus replication fitness and decrease pandemic potential [Genetic Diversity and Evolution]

  • H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T), 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in the field isolates in 2019. To investigate the potential threat of the serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in HA protein might pose a reduced pandemic risk but remain a heightened threat for poultry.

    IMPORTANCE

    Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in the virus-specific antiserum give rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunised or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chick embryo as well as (ii) increased acid and thermal stability, but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing indicated mutations remain a significant risk to the poultry due to antigenic drift and improved fitness for poultry.

  • Herpes Simplex Virus Type 2 Counteracts Neurite Outgrowth Repulsion During Infection In A Nerve Growth Factor-Dependent Manner [Virus-Cell Interactions]

  • During primary infection, herpes simplex virus type 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors, attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesised that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis we exposed sensory neurons to nerve growth factor (NGF) and mock or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secreted factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduced this repelling phenotype, increasing neurite numbers. The HSV-2 mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection had not been investigated. Therefore, we addressed whether gG2 contributed to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of non-neuronal cells reduced their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2 mediated pathogenesis.

    Importance: Herpes simplex virus type 2 (HSV-2) is a prevalent human pathogen that establishes life-long latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that non-neuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition, and rather stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.

  • Regional brain recovery from acute synaptic injury in SIV-infected rhesus macaques associates with heme oxygenase isoform expression [Pathogenesis and Immunity]

  • Brain injury occurs within days in SIV or HIV infection and some recovery may occur within weeks. Inflammation and oxidative stress associate with such injury, but what drives recovery is unknown. Chronic HIV infection associates with reduced brain frontal cortex expression of the antioxidant/anti-inflammatory enzyme heme oxygenase-1 (HO-1) and increased neuroinflammation in individuals with cognitive impairment. We hypothesized that acute regional brain injury and recovery associate with differences in regional brain HO-1 expression. Using SIV-infected rhesus macaques, we analyzed multiple brain regions through acute and chronic infection (90 dpi), and quantified markers: viral (SIV gag RNA), synaptic (PSD-95, synaptophysin), axonal (neurofilament/NFL), inflammatory, and antioxidant (enzymes, including heme oxygenase isoforms [HO-1, HO-2]). PSD-95 was reduced in brainstem, basal ganglia, neocortex, and cerebellum within 13 dpi, indicating acute synaptic injury throughout the brain. All areas except brainstem recovered. Unchanged NFL is consistent with no acute axonal injury. SIV RNA expression was highest in brainstem throughout infection, and it associated with neuroinflammation. Surprisingly, during synaptic injury and recovery phases, HO-2, and not HO-1, progressively decreased in brainstem. Thus, acute SIV synaptic injury occurs throughout the brain, with spontaneous recovery in regions other than brainstem. Within the brainstem, the high SIV load and inflammation, along with reduction of HO-2 may impair recovery. In other brain regions, stable HO-2 expression with or without increasing HO-1 may promote recovery. Our data support roles for heme oxygenase isoforms in modulating recovery from synaptic injury in SIV infection and suggest their therapeutic targeting for promoting neuronal recovery.

    Importance Brain injury induced by acute simian (or human) immunodeficiency virus infection may persist or spontaneously resolve in different brain regions. Identifying the host factor (s) that promote spontaneous recovery from such injury may reveal targets for therapeutic drug strategies for promoting recovery from acute neuronal injury. The gradual recovery from the injury observed in many, but not all, brain regions in the rhesus macaque model is consistent with there being a possible therapeutic window of opportunity for intervening to promote recovery, even in those regions not showing spontaneous recovery. In persons living with human immunodeficiency virus infection, such neuroprotective treatments could ultimately be considered as adjuncts to initiation of antiretroviral drug therapy.

  • Inactivation of genes by frameshift mutations provides rapid adaptation of an attenuated vaccinia virus [Genetic Diversity and Evolution]

  • Unlike RNA viruses, most DNA viruses replicate their genomes with high fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies reveal rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way adaptation can occur is by an accordion mechanism in which gene copy number increases followed by base substitutions and finally contraction of gene copy number. Here we show rapid acquisition of multiple adaptive mutations by a gene inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During ten passage rounds, the attenuated VACV was replaced by the mutated strains. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely A26L, G6R and A14.5L, achieved 74 to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L, G6R or both deleted, which increased virus replication and decreased particle/plaque forming unit ratios.

    IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.

  • A Novel Mucosal Adjuvant System for the Immunization Against Avian Coronavirus Causing Infectious Bronchitis. [Vaccines and Antiviral Agents]

  • Infectious Bronchitis (IB) caused by Infectious Bronchitis Virus (IBV) is currently a major threat to chicken health with multiple outbreaks being reported in the US over the past decade. Modified live virus (MLV) vaccines used in the field can persist and provide the genetic material needed for recombination and emergence of novel IBV serotypes. Inactivated and subunit vaccines overcome some of the limitations of MLV with no risk of virulence reversion and emergence of new virulent serotypes. However, these vaccines are weakly immunogenic and poorly protective. There is an urgent need to develop more effective vaccines that can elicit a robust, long-lasting immune response. In this study, we evaluate a novel adjuvant system developed from Quil-A and chitosan (QAC) for the intranasal delivery of nucleic acid immunogens to improve protective efficacy. The QAC adjuvant system forms nanocarriers (llt;100 nm) that efficiently encapsulate nucleic acid cargo, exhibit sustained release of payload and can stably transfect cells. Encapsulation of plasmid DNA vaccine expressing IBV nucleocapsid (N) protein by the QAC adjuvant system (pQAC-N) enhanced immunogenicity as evidenced by robust induction of adaptive humoral and cellular immune responses post vaccination and challenge. Birds immunized with pQAC-N showed reduced clinical severity and viral shedding post challenge on par with protection observed with current commercial vaccines without the associated safety concerns. Presented results indicate that the QAC adjuvant system can offer a safer alternative to the use of live vaccines against avian and other emerging coronaviruses.

    Importance According to the 2017 US agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67 percent of the industry value with the production of ggt; 50 billion pounds of chicken meats. The economic success of the poultry industry in the USA hinges on the extensive use of vaccines to control Infectious Bronchitis Virus (IBV) and other poultry pathogens. Majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could potentially revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.

  • Development of reverse genetics for the prototype New World mammarenavirus Tacaribe virus [Vaccines and Antiviral Agents]

  • The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease but it has been shown to protect against Argentine hemorrhagic fever like disease in marmosets infected with the New World mammarenavirus Juniiacute;n virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant (r)TCRV. We successfully rescued a wild-type (WT) rTCRV, a tri-segmented form of TCRV expressing two reporter genes (r3TCRV); and a bi-segmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV, and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39 nucleotide (nt) deletion (39) in the non-coding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, a rTCRV containing this 39 nt deletion in the L-IGR (rTCRV/39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or a vaccine vectors.

    IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin that is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is non-pathogenic in humans but able to provide protection against Juniiacute;n virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially, other viral infections. Here we describe, for the first time, the feasibility to generate recombinant (r)TCRV using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

  • Co-evolution of AAV capsid antigenicity and tropism through a structure-guided approach [Gene Delivery]

  • Adeno-associated viruses (AAV) are composed of non-enveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone, but requires harnessing the 3D structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper Adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying non-natural surface loops that displays simultaneously tropism for human hepatocytes, increased gene transfer efficiency and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species compared to AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties such as AAV capsid antibody evasion and tropism can be co-evolved by combining rational design and library-based evolution for clinical gene therapy.

    Importance Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone, but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to co-evolve multiple, desirable properties onto AAV by harnessing 3D structural information.

  • Single point mutations within the Coxsackie B virus receptor-binding site promote resistance against soluble virus receptor traps [Vaccines and Antiviral Agents]

  • Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand-foot-mouth disease to life-threatening conditions such as pancreatitis, myocarditis and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc result in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3M) and one with partial (CVB3K) resistance. DNA sequence alignment of the resistant virus variant CVB3M with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus's interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3N2139S to sCAR-Fc. Interestingly, development of resistance by CVB3-H3N2139S and the exemplarily investigated CVB3M-clone 2 (CVB3M2) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and the viral replication in comparison to the parental strains.

    These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single amino acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness.

    IMPORTANCE The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, thus representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules.

  • Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication [Virus-Cell Interactions]

  • Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and largely localized to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 pro-inflammatory and/or angiogenic viral proteins, in HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), in addition to an MCD-related disorder involving systemic elevation of pro-inflammatory cytokines, including vIL-6 and human IL-6 (hIL-6). In these diseases, lytic (productive) replication, in addition to viral latency, is believed to play a critical role. Pro-replication activity of vIL-6 has been identified experimentally in PEL and endothelial cells, but the relative contributions of different vIL-6 interactions have not been established. Productive interactions of vIL-6 with the IL-6 signal transducer, gp130, can occur within the ER, but vIL-6 also interacts in the ER with a non-signaling receptor called vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2), calnexin, and VKORC1v2- and calnexin-associated proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII). Here, we report the systematic characterization of interaction-altered vIL-6 variants and the lytic phenotypes of recombinant viruses expressing selected variants. Our data identify the critical importance of vIL-6 and its ER-localized activity via gp130 to productive replication in iSLK (inducible epithelial) cells, absence of detectable involvement of vIL-6 interactions with VKORC1v2, GlucII or UGGT1, and the insufficiency and lack of direct contributory effects of extracellular signaling by vIL-6 or hIL-6. These findings, obtained through genetics-based approaches, complement and extend previous analyses of vIL-6 activity.

    IMPORTANCE Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) was the first viral IL-6 homologue to be identified. Experimental and clinical evidence suggests that vIL-6 is important for the onset and/or progression of HHV-8-associated endothelial- and B-cell pathologies, including AIDS-associated Kaposi's sarcoma and multicentric Castleman's disease. The protein is unusual in its poor secretion from cells and its intracellular activity; it interacts, directly or indirectly, with a number of proteins beyond the IL-6 signal transducer, gp130, and can mediate activities through these interactions in the endoplasmic reticulum. Here, we characterize with respect to protein interactions and signal transducing activity a panel of vIL-6 variants and utilize HHV-8 mutant viruses expressing selected variants in phenotypic analyses. Our findings establish the importance of vIL-6 in HHV-8 productive replication and the contributions of individual vIL-6-protein interactions to HHV-8 lytic biology. This work furthers understanding of the biological significance of vIL-6 and its unique intracellular interactions.

  • SUMOylation targets AAV capsids but mainly restricts transduction by cellular mechanisms [Virus-Cell Interactions]

  • Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy, due to its non-pathogenic nature, ease of production and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the Small Ubiquitin-like Modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated as indicated by deletion and point mutations of VP2 or the obstruction of its N-terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability compared to non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation, however, it reduced activation of gene transduction by shut down of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor, that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the co-expression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.

    Importance

    Host factors within the cell are the major mode of restriction of Adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide siRNA screen, we identified that proteins of the Small Ubiquitin-like Modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV virus directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

  • A rare mutation in an infant derived HIV-1 envelope glycoprotein alters interprotomer stability and susceptibility to broadly neutralizing antibodies targeting the trimer apex. [Genetic Diversity and Evolution]

  • The envelope glycoprotein (Env) of human immunodeficiency virus-1 (HIV-1) is the sole target of broadly neutralizing antibodies (bnAbs). Several mechanisms, such as acquisition of mutations, variability of loop length and alterations in glycan pattern are employed by the virus to shield neutralizing epitopes on the env, to sustain survival and infectivity within the host. Identification of mutations that lead to viral evasion from host immune response is essential for optimization and engineering of Env-based trimeric immunogens. Herein, we report a rare leucine-to-phenylalanine escape mutation (L184F) at the base of hypervariable loop 2 (population frequency of 0.0045%) in a nine-month-old perinatally HIV-1 infected infant broad neutralizer. The L184F mutation altered the trimer conformation by modulating intramolecular interactions stabilizing the trimer apex and led to viral escape from autologous plasma bnAbs and known N160 glycan targeted bnAbs. The L184F amino acid change led to acquisition of a relatively open trimeric conformation, often associated with tier 1 HIV-1 isolates and an increased susceptibility to neutralization by polyclonal plasma antibodies of weak neutralizers. While there was no impact of the L184F mutation on free virus transmission, a reduction in cell-to-cell transmission was observed. In conclusion, we report a naturally selected viral mutation L184F that influenced a change in conformation of the Env trimer apex as an escape mechanism from contemporaneous plasma V2 apex targeted nAbs. Further studies should be undertaken to define viral mutations acquired during natural infection, to escape selection pressure exerted by bnAbs, to inform vaccine design and bnAb based therapeutic strategies.

    Importance Design of HIV-1 envelope-based immunogens, capable of eliciting broadly neutralizing antibodies (bnAbs), are currently under active research. Some of the most potent bnAbs target the quaternary epitope at the V2 apex of HIV-1 Env trimer. By studying naturally circulating viruses from an HIV-1 perinatally infected infant, with plasma neutralizing antibodies targeted to the V2-apex, we identified a rare leucine to phenylalanine substitution, in two out of six functional viral clones, that destabilized the trimer apex. This single amino acid alteration impaired the interprotomeric interactions that stabilize the trimer apex, resulting in an open trimer conformation, and escape from broadly neutralizing autologous plasma antibodies and known V2-apex directed bnAbs, thereby favouring viral evasion of the early bnAb response of the infected host. Defining the mechanisms by which naturally occurring viral mutations influence the sensitivity of HIV-1 to bnAbs will provide information for development of vaccines and bnAbs as anti-HIV-1 reagents.

  • Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion [Transformation and Oncogenesis]

  • Merkel cell polyomavirus (MCV) small T antigen (sT) is the main oncoprotein for the development of Merkel cell carcinoma (MCC). MCC is a rare, clinically aggressive neuroendocrine tumor of the skin with a high propensity for local, regional, and distant spread. The dysregulation of matrix metalloproteinase-9 (MMP-9) has been implicated in multiple essential roles in the development of various malignant tumor cell invasion and metastasis. Previously, MCV sT was shown to induce the migratory and invasive phenotype of MCC cells through the transcriptional activation of the sheddase molecule, ADAM 10 (A disintegrin and metalloprotease domain-containing protein 10). In this study, we show that MCV sT protein stimulates differential expression of epithelialnndash;mesenchymal transition (EMT)-associated genes, including MMP-9 and Snail. This effect is dependent on the presence of the large T stabilization domain (LSD), which is known to be responsible for cell transformation through targeting of promiscuous E3 ligases, including FBW7, a known MMP-9 and Snail regulator. Chemical treatments of MMP-9 markedly inhibited MCV sT-induced cell migration and invasion. These results suggest that MCV sT contributes to the activation of MMP-9 as a result of FBW7 targeting, and increases the invasive potential of cells, which can be used for targeted therapeutic intervention.

    IMPORTANCE Merkel cell carcinoma (MCC) is the most aggressive cutaneous tumor without clearly defined treatment. Although MCC has a high propensity for metastasis, little is known about the underlying mechanisms that drive MCC invasion and metastatic progression. MMP-9 has shown to play a detrimental role in many metastatic human cancers, including melanoma and other non-melanoma skin cancers. Our study shows that MCV sT-mediated MMP-9 activation is driven through the LSD, a known E3 ligase targeting domain, in MCC. MMP-9 may serve as the biochemical culprit to target and develop a novel approach for the treatment of metastatic MCC.

  • Combination of CD8{beta} depletion and IL-15 superagonist N-803 induces virus reactivation in SHIV-infected, long-term ART-treated rhesus macaques [Pathogenesis and Immunity]

  • The "shock and kill" strategy predicates that virus reactivation in latently infected cells is required to eliminate the HIV reservoir. In a recent study, we show robust and persistent induction of plasma viremia in ART-treated SIV-infected rhesus macaques (RMs) undergoing CD8aalpha; depletion and treated with the IL-15 superagonist N-803 (McBrien, J.B. et al. Nature 578, 154nndash;159 (2020), https://doi.org/10.1038/s41586-020-1946-0). Of note, in the study we used an antibody targeting CD8aalpha;, therefore depleting NK cells, NKT cells, and T cells in addition to CD8+ T-cells. In the current proof-of-concept study, we tested whether virus reactivation can be induced by administration of N-803 to SHIV-infected, ART-treated RMs that are selectively depleted of CD8+ T cells via the CD8bbeta;-targeting antibody CD8b255R1. CD8bbeta; depletion was performed in five SHIVSF162P3-infected RMs treated with ART for 12 months and with plasma viremia consistently below 3 copies/ml. All animals received four weekly doses of N-803 starting at the time of CD8b255R1 administration. Induction of detectable plasma viremia was observed in three out of five RMs, with the level of virus reactivation seemingly correlated to the frequency of CD8+ T-cells following CD8bbeta; depletion as well as the level of virus reactivation observed when the same animals underwent CD8aalpha; depletion and N-803 administration after 24 weeks of ART. These data indicate that CD8bbeta; depletion and N-803 administration can induce virus reactivation in SHIVSF162P3-infected RMs despite suboptimal depletion of CD8+ T cells and profound ART-induced suppression of virus replication, thus confirming a critical role for these cells in suppressing virus production and/or reactivation in vivo under ART.

    Importance The "shock and kill" HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to impact on the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our lab to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8+ lymphocytes in suppressing the latency-reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8bbeta;-targeting antibody, which would specifically deplete CD8+ T cells, would yield similar levels of virus reactivation. We observed induction of plasma viremia which correlated with the efficacy of the CD8 depletion strategy.

  • Characterization of a novel alphaherpesvirus isolated from the fruit bat Pteropus lylei, in Viet Nam [Genetic Diversity and Evolution]

  • Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)", was isolated from urine of the fruit bat Pteropus lylei in Viet Nam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to code 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblast cells than HSV-1 did. PLAHV was serologically related with another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.

    IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Viet Nam. The whole genome sequence was determined and was predicted to code 72 open reading frames in the 144,008-bp virus genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

  • Metabolic Syndrome and Viral Pathogenesis: Lessons from Influenza and Coronaviruses [Gem]

  • Metabolic syndrome increases the risk of severe disease due to viral infection. Yet, few6 studies have assessed the pathogenesis of respiratory viruses in high-risk populations. Here, we summarize how metabolic dysregulation impairs immune responses and we define the role of metabolism during influenza and coronavirus infections. We also discuss the use of various in vitro, in vivo, and ex vivo models to elucidate the contribution of host factors to viral susceptibility, immunity, and disease severity.

  • Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates Both Early and Late Steps in the Membrane Fusion Cascade [Virus-Cell Interactions]

  • Medically important paramyxoviruses such as measles, mumps, parainfluenza, Nipah, and Hendra viruses infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytia formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. Here, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its pre-fusion and post-fusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion-pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV, and likely for the related Henipavirus genus and possibly Paramyxoviridae family members.

    IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens such as measles, mumps, parainfluenza, and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the CNS and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40-100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. Here, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets the HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

  • RNA-seq transcriptome analysis reveals LTR-retrotransposons modulation in Human Peripheral Blood Mononuclear Cells (PBMCs) after in vivo Lipopolysaccharides (LPS) injection [Virus-Cell Interactions]

  • Human Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) are retroviral sequences that integrated into the germline cells millions years ago. Transcripts of these LTR-retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. In this work, we focused on the HERVs/MaLRs expression and modulation in a scenario of immune system activation. We used a public dataset of Human Peripheral Blood Mononuclear Cells (PBMCs) RNA-seq from 15 healthy participants to a clinical trial before and after the exposure to Lipopolysaccharide (LPS), for which we established an RNA-seq workflow for the identification of expressed and modulated cellular genes and LTR-retrotransposon elements.

    IMPORTANCE We described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4 % of the LTR-retrotransposons loci were expressed, and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERVs and MaLRs loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci differentially expressed, checking their genomic context of insertion and observing a general co-localization with genes that are involved and modulated in the immune response, as consequence of LPS stimulation. The analyses of HERV and MaLR expression and modulation show that these LTR-retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR-retrotransposons and the immune host response.

  • Nonhuman Primate Testing of the Impact of Different Treg Depletion Strategies on Reactivation and Clearance of Latent Simian Immunodeficiency Virus [Pathogenesis and Immunity]

  • Regulatory T cells (Tregs) may be key contributors to the HIV/SIV latent reservoir, as they harbor high levels of HIV/SIV; reverse CD4+ T cell immune activation status, increasing the pool of resting CD4+ T cells; impair CD8+ T cell function, favoring HIV persistence. We tested the hypothesis that Treg depletion is a valid intervention towards an HIV cure by depleted Tregs in fourteen SIVsab-infected RM controllers through different strategies: administration of an anti-CCR4 immunotoxin; two doses of an anti-CD25 immunotoxin (IL-2 with diphtheria toxin: IL-2-DT); or two combinations of both. All these treatments resulted in significant depletion of the circulating Tregs (ggt;70%) and their partial depletion in the gut (25%) and lymph nodes (ggt;50%). The fractions of CD4+ T cells expressing Ki-67 increased up-to 80% in experiments containing IL-2-DT and only 30% in anti-CCR4nndash;treated RMs, paralleled by increases in the inflammatory cytokines. In the absence of ART, plasma virus rebounded to 103 vRNA copies/mL by day 10 post-IL-2-DT administration. A large but transient boost of the SIV-specific CD8+ T cell responses occurred in IL-2-DTnndash;treated RMs. Such increases were minimal in the RMs receiving anti-CCR4-based regimens. Five RMs received IL-2DT on ART, but treatment was discontinued because of high toxicity and lymphopenia. As such, while all treatments depleted a significant proportion of Tregs, the side effects in the presence of ART prevent their clinical use and call for different Treg depletion approaches. Thus, based on our data, Treg targeting as a strategy for HIV cure cannot be discarded.

    IMPORTANCE Regulatory T cells (Tregs) can decisively contribute to the establishment and persistence of the HIV reservoir, as they harbor high levels of HIV/SIV, increase the pool of resting CD4+ T cells by reversing their immune activation status, and impair CD8+ T cell function, favoring HIV persistence. We tested multiple Treg depletion strategies and showed that all of them are at least partially successful in depleting Tregs. As such, Treg depletion appears to be a valid intervention towards an HIV cure, reducing the size of the reservoir, reactivating the virus, and boosting cell-mediated immune responses. Yet, when Treg depletion was attempted in ART-suppressed animals, the treatment had to be discontinued due to high toxicity and lymphopenia. Therefore, while Treg targeting as a strategy for HIV cure cannot be discarded, the methodology for Treg depletion has to be revisited.

  • A stronger transcription regulatory circuit of HIV-1C drives the rapid establishment of latency with implications for the direct involvement of Tat [Virus-Cell Interactions]

  • The magnitude of transcription factor binding site variation emerging in HIV-1C, especially the addition of NF-B motifs by sequence duplication, makes the examination of transcriptional silence challenging. How can HIV-1 establish and maintain latency despite having a strong LTR? We constructed panels of sub-genomic reporter viral vectors with varying copy numbers of NF-B motifs (0 to 4 copies) and examined the profile of latency establishment in Jurkat cells. We found surprisingly that the stronger the viral promoter, the faster the latency establishment. Importantly, at the time of commitment to latency and subsequent points, Tat levels in the cell were not limiting. Using highly sensitive strategies, we demonstrate the presence of Tat in the latent cell, recruited to the latent LTR. Our data allude, for the first time, to Tat establishing a negative feedback loop during the late phases of viral infection, leading to the rapid silencing of the viral promoter.

    Importance Over the past 10-15 years, HIV-1C has been evolving rapidly towards gaining stronger transcriptional activity by sequence duplication of major transcription factor binding sites. The duplication of NF-B motifs is unique and exclusive for HIV-1C, a property not shared with any of the other eight HIV-1 genetic families. What mechanism(s) does HIV-1C employ to establish and maintain transcriptional silence despite the presence of a strong promoter and a concomitant strong, positive transcriptional feedback is the primary question we attempted to address in the present manuscript. The role Tat plays in latency reversal is well established. Our work with the most common HIV-1 subtype C (HIV-1C) offers crucial leads towards Tat possessing a dual-role in serving both as transcriptional activator and repressor at different phases of the viral infection of the cell. The leads we offer through the present work have significant implications for HIV-1 cure research.

  • The SARS-CoV-2 N protein is a good component in a vaccine [Letters to the Editor]

  • Dutta and co-workers suggest in a recent letter (1) that the SARS-CoV-2 nucleoprotein (N) might be a good vaccine target....

  • Broad and differential animal ACE2 receptor usage by SARS-CoV-2 [Virus-Cell Interactions]

  • The COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.

    IMPORTANCE SARS-CoV-2 uses human ACE2 as primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologues and found that wild type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models and molecular basis of receptor binding for SARS-CoV-2.

  • Cell fusion induced by a fusion-active form of human cytomegalovirus glycoprotein B is inhibited by antibodies directed at AD-5 in the ectodomain of gB [Vaccines and Antiviral Agents]

  • Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero. Virus neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is non-fusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to related class III fusion proteins including Vesicular Stomatitis Virus glycoprotein G or Baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus neutralizing gB-specific monoclonal antibodies (mAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing mAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB mAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.

    IMPORTANCE HCMV is a major global health concern and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence is often included as a component of intervention strategies. By generation of fusion-active gB chimeras we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.

  • H9N2 influenza virus infections in human cells require a balance between NA sialidase activity and HA receptor affinity [Virus-Cell Interactions]

  • Some avian influenza (AI) viruses have a deletion of up to 20-30 amino acids in their NA stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses only have a 2 or 3 amino acid deletion, which were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8 amino acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses with various NA stalk lengths and were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low molecular weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with NA stalk length, but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry in host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with an HA with higher aalpha;2,6 Sia affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicated that that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, was required for optimal G1 virus entry into human airway cells.

    IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage most prevalent worldwide. In this study, we isolated G1 variants carrying an 8 amino acid deletion in their NA stalk, which was, to our knowledge, the longest deletion found in H9N2 viruses in the field. NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species-specific and dependent on the corresponding HA binding affinity. Our results suggested that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.

  • Swine Promyelocytic Leukemia Isoform II Inhibits Pseudorabies Virus Infection by Suppressing Viral Gene Transcription in PML-NBs [Cellular Response to Infection]

  • Promyelocytic leukaemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against aalpha;-herpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to aalpha;-herpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoform sPML-II/IIa, not sPML-I and IVa, expressed in a sPML knockout cells inhibits PRV infection. Both the unique 7b region of sPML-II and sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residue 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.

    IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of aalpha;-herpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C-terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an anti-viral function, but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.

  • Sp3 transcription factor cooperates with the Kaposi's sarcoma-associated herpesvirus ORF50 protein to synergistically activate specific viral and cellular gene promoters [Genome Replication and Regulation of Viral Gene Expression]

  • Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded ORF50 protein is the key transactivator responsible for the latent-to-lytic switch. Herein, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50, and successfully identified an ORF50-responsive element located in the promoter region between nndash;97 and nndash;44 (designated as 56p-RE). This 56p-RE element contains a non-canonical RBP-J-binding sequence and a non-consensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-J, Sp3 and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient reporter analysis showed that Sp3, but not RBP-J or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searching revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular IL-10 contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could be also synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, the transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57 and K6 promoters. Collectively, our study demonstrates that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for the transcriptional synergy.

    IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. We here find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase) and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in the ORF50-mediated transactivation, and propose a new subclass of the ORF50-responsive elements in the viral and cellular promoters.

  • Genomic Analysis of Early SARS-CoV-2 Variants Introduced in Mexico [Genetic Diversity and Evolution]

  • The COVID-19 pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to implement effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, the SARS-CoV-2 lineage A/G, containing mostly sequences from North America, and the lineage B/S containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized fourteen independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represent local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described for the country. Within this local transmission cluster, we also identified the H49Y amino acid change in the Spike protein. This mutation is a homoplasy occurring independently through time and space, and may function as a molecular marker to follow on any further spread of these viral variants throughout the country. Our results depict the general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts.

    IMPORTANCE Understanding the introduction, spread and establishment of SARS-CoV-2 within distinct human populations is crucial to implement effective control strategies as well as the evolution of the pandemics. In this work, we describe that the initial virus strains introduced in Mexico came from Europe and the United States and the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains having the mutation H49Y in the Spike protein, that could be further used as a molecular marker to follow viral spread within the country and the region.

  • Subclinical CMV and EBV Shedding is associated with increasing HIV DNA Molecular Diversity in Peripheral Blood during Suppressive Antiretroviral Therapy [Genetic Diversity and Evolution]

  • Cytomegalovirus (CMV) almost universally infects persons with HIV (PWH), and it is a driver of persistent inflammation and HIV persistence. The mechanisms underlying the association between CMV (and possibly other herpes viruses) and HIV persistence are unclear. Serially collected blood samples were obtained from men who have sex with men (MSM) who started ART within 1 year of their estimated date of HIV infection (EDI). Total CMV and EBV DNA were quantified in peripheral blood mononuclear cells by droplet digital PCR. Deep sequencing of HIV DNA partial env gene was performed and the dynamics of viral diversity over time was analyzed in relation to CMV and EBV shedding status. In total, 37 MSM PWH were included and followed for a median of 23 months (IQR: 22-28). Participants started ART within a median of 3.1 months (IQR: 1.5-6.5) after EDI and remained virally suppressed thereafter. Eighteen participants (48.6%) were classified as high EBV shedders, while 19 (51.4%) were classified as CMV shedders. In multivariate analyses, normalized molecular diversity levels tended to increase over time among participants with detectable CMV and high EBV DNA (0.03pplusmn;0.02, p=0.08), while they significantly declined among participants with no/low viral shedding (-0.04pplusmn;0.02, p=0.047, Interaction pllt;0.01). Subclinical CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART. Whether persistent viral replication could be targeted as a strategy to reduce the size of the latent HIV reservoir is an avenue that should be explored.

    IMPORTANCE As part of this study we evaluated the molecular characteristics of the HIV DNA reservoir over time during antiretroviral treatment (ART) in relation to other chronic viral infections (i.e. Cytomegalovirus [CMV] and Epstein-Barr Virus [EBV]). We demonstrated that presence of CMV and high-level EBV DNA in peripheral blood cells was associated with changes in HIV DNA molecular diversity. Specifically, HIV DNA molecular diversity increased over time among participants with detectable CMV and high-level EBV DNA, while it significantly declined among participants with no/low viral shedding. Although the current study design does not allow causality to be inferred, it does support the theory that persistent CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART even when ART is initiated during the earliest phases of HIV infection.

  • PD-L1 checkpoint inhibition narrows the antigen-specific T cell receptor repertoire in chronic LCMV infection [Cellular Response to Infection]

  • Checkpoint inhibitors are effective in restoring exhausted CD8+ T cell responses in persistent viral infections or tumors. Several compounds are in clinical use for different malignancies but also trials in patients with chronic viral infections were conducted. In the mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, it was shown that checkpoint inhibitor treatment increased T cell proliferation and functionality but its influence on the antigen-specific T cell receptor (TCR) repertoire is unknown.

    NP396-specific CD8+ T cells dominate during acute LCMV infection and are predominantly exhausted during chronic infection. Next generation sequencing of NP396-specific TCRs showed that exhaustion corresponds with a significantly reduced NP396-specific TCR repertoire diversity: Shannon Index of 4 in immunized to 2.6 in persistently infected mice. Anti-PD-L1 treatment during persistent LCMV infection restored NP396-specific T cell responses and reduced viral titers. Nevertheless, "aalpha;PD-L1-treated" mice showed an even more narrowed TCR repertoire, with reduced TCR diversity compared to persistently infected control mice (Shannon Index of 2.1 and 2.6, respectively). Interestingly, aalpha;PD-L1 treatment induced narrowing of the TCR repertoire negatively correlates with functional and physical restoration of the antigen-specific T cell response. Further, we found that private, hyper-expanded TCR clonotypes dominated the T cell response after aalpha;PD-L1 treatment. Although being private, these top clonotypes from "aalpha;PD-L1-treated" mice revealed a more "closely-related" CDR3 motif pattern, compared to top clonotypes from persistently infected control mice. In conclusion, although targeting the PD-1/PD-L1 pathway re-invigorates exhausted CD8+ T cells, it fails to restore T-cell repertoire diversity.

    IMPORTANCE Checkpoint inhibitors are effective immunotherapeutics to restore cancer- and virus-induced exhausted CD8+ T cells, by enhancing the quality and survival of immune responses. Although checkpoint inhibitors are already used as therapy against various cancers, not much is known about their multifaceted impact on the exhausted CD8+ T cell receptor (TCR) repertoire. This study describes for the first time the evolvement of an exhausted antigen-specific CD8+ TCR repertoire under checkpoint inhibitor treatment. By using a well-established virus model, we were able to show major shifts towards oligoclonality of the CD8+ TCR repertoire response against a massively exhausted lymphocytic choriomeningitis virus (LCMV) epitope. While supporting viral control in the LCMV model, oligoclonality and more private of TCR repertoires may impact future pathogenic challenges and may promote viral escape. Our results may explain the ongoing problems of viral escapes, unpredictable autoimmunity and heterogeneous responses appearing as adverse effects of checkpoint inhibitor treatments.

  • Reactivation of Epstein-Barr Virus By HIF-1{alpha} Requires p53 [Genome Replication and Regulation of Viral Gene Expression]

  • We previously reported that the cellular transcription factor, HIF-1aalpha;, binds a hypoxia-response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. Here, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1aalpha;-stabilizing drugs: iron chelator [Desferalrreg; (DFO,)]; neddylation inhibitor [Pevonedistatrreg; (MLN-4924)]; and prolyl hydroxylase inhibitor [Roxadustatrreg; (FG-4592)]. DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1aalpha; and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with shRNAs. Likewise, HIF-1aalpha; failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1aalpha; to Zp in ChIP assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1aalpha; requires phosphorylated, wild-type p53 as a co-activator, with HIF-1aalpha; binding recruiting p53 to Zp.

    IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1aalpha; binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1aalpha;-stabilizing drugs can induce EBV reactivation. Here, we determined which EBV-positive cell lines are reactivated by classes of HIF-1aalpha;-stabilizing drugs. We found, unexpectedly, that HIF-1aalpha;-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as Nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1aalpha;-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

  • Inhibition of human adenovirus replication by the importin {alpha}/{beta}1 nuclear import inhibitor ivermectin [Vaccines and Antiviral Agents]

  • Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease, however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-aalpha; (Imp-aalpha;) and -bbeta;1 (Imp-bbeta;1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-aalpha; without affecting the interaction between Imp-aalpha; and Imp-bbeta;1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-aalpha;/bbeta;1 mediated nuclear import.

    IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms, however individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro. This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of Imp-aalpha; to recognize nuclear localization sequences, without effecting the Imp-aalpha;/bbeta;1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

  • Serpentoviruses, more than respiratory pathogens [Pathogenesis and Immunity]

  • In recent years nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full post mortem examinations on 30 serpentovirus RT-PCR positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus not only infects a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts etc.), but also intravascular monocytes, intralesional macrophages and endothelial cells. By next-generation sequencing we obtained full length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection associated respiratory or systemic disease did not reveal sequence association to phenotypes, however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.

    IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that compromise important zoonotic pathogens such as MERS-CoV, SARS-CoV, and SARS-CoV-2. Serpentoviruses belong to the same order as the mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order nidovirales.

  • LY6E Restricts the Entry of Human Coronaviruses, Including the Currently Pandemic SARS-CoV-2 [Virus-Cell Interactions]

  • C3A is a sub-clone of human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT) and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins with identified function of interfering virus entry, expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFITM3 restriction of human coronavirus entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.

    IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control by host innate and adaptive immune responses. In the last decade, several interferon inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as host factors to facilitate the entry of several human pathogenic viruses, including human immunodeficiency virus, influenza A virus and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.

  • Reconstitution of an RNA virus replicase in artificial giant unilamellar vesicles supports full replication and provides protection for the dsRNA replication intermediate [Genome Replication and Regulation of Viral Gene Expression]

  • Positive-strand RNA viruses are important pathogens of humans, animals and plants and replicate inside host cells by co-opting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and co-opted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with ER-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the dsRNA replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by PI (3)P and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 co-opted replicase assembly factors, Vps34 PI3K and the membrane-bending ESCRT factors are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential co-opted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.

    Significance: Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and co-opt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, the authors have developed artificial giant unilamellar vesicles (GUVs)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential co-opted cellular factors.

  • Murine leukemia virus P50 protein counteracts APOBEC3 by blocking its packaging [Cellular Response to Infection]

  • Apolipoprotein B editing, catalytic subunit 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like HIV-1 encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C-terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout and wild type mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo. Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than WT viruses and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.

    IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus when compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

  • Phospho-regulation of a conserved herpesvirus tegument protein by a virally-encoded protein kinase in viral pathogenicity and potential linkage between its evolution and viral phylogeny [Genetic Diversity and Evolution]

  • Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multi-functional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein UL7 as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo. Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7 of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestral UL7 for HSV-1, HSV-2, and ChHV, and UL7s of other members of the genus Simplexvirus that are phylogenically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplexviruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1 had no effect on viral replication and pathogenicity in mice in contrast to phosphorylation in HSV-2. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phospho-regulation of UL7 during their evolution because the phospho-regulation had an impact on viral fitness in vivo, whereas most other simplexviruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivo.

    IMPORTANCE It has been hypothesized that the evolution of protein phospho-regulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phospho-regulation and phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phospho-regulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo. We also showed that evolution of Us3 phospho-regulation of UL7 was linked to phylogeny of viruses that are phylogenically close to HSV-2 and to phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have or have not evolved phospho-regulation, respectively. This study reports the first evidence showing that evolution of viral phospho-regulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phospho-regulation during viral evolution.

  • Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element [Genome Replication and Regulation of Viral Gene Expression]

  • The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently-developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein, in addition to the 12 viral proteins. To address this, we replaced the NSP3 open-reading-frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing, generally genetically stable, and expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses may prove useful as expression vectors.

    Importance. Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein, in addition to the full complement of viral proteins. Through this approach, we have generated wildtype-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live-cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as vaccine expression vector.

  • Fiber-1, not Fiber-2, directly mediates the infection of the pathogenic serotype 4 fowl adenovirus via its shaft and knob domains [Virus-Cell Interactions]

  • Recently, the disease of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) has spread widely and resulted in huge economic loss to poultry industry. Although the genome of FAdV-4 has two fiber genes (Fiber-1 and Fiber-2), the exact role of the genes in the infection of FAdV-4 is barely known. In this study, through superinfection resistant analysis and interfering assay, we found that Fiber-1, but not Fiber-2, was the key factor for directly triggering the infection of FAdV-4. The truncation analysis further revealed that both of the shaft and knob domains of Fiber-1 were required for the infection. Moreover, the sera against the knob domain could block FAdV-4 infection, and the knob-containing fusion protein could provide efficient protection against the lethal challenge of FAdV-4 in chickens. All the data demonstrated the significant roles of Fiber-1 and its knob domain in directly mediating the infection of FAdV-4, which established a foundation for identifying the receptor of FAdV-4 and developing efficient vaccines against FAdV-4.

    Importance: Among 12 serotypes of FAdV, FAdV-1, FAdV-4 and FAdV-10 all carry two Fiber genes (i.e., Fiber-1 and Fiber-2) whereas other serotypes have only one. As important viral surface proteins, the Fibers play a vital role in the infection and pathogenesis of FAdV. However, the importance of the Fibers to the infection and pathogenesis of FAdV may be different from each other. Recent studies reveal that Fiber-2 is identified as a determinant of virulence, but which Fiber triggers the infection of FAdV-4 almost remains unknown In this study, Fiber-1 was identified as a key factor for directly mediating the infection of FAdV-4 through its shaft and knob domains, whereas Fiber-2 did not play roles in triggering FAdV-4 infection. The results suggest that Fiber-1 and its knob domain may serve a target for identifying the receptor of FAdV-4 and developing efficient drugs or vaccines against FAdV-4.

  • Dimerization of dengue virus E-subunits impacts antibody function and domain focus. [Vaccines and Antiviral Agents]

  • Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live-vaccines, tetravalent DENV envelope (E)-protein subunit vaccines are likely to stimulate balanced immune responses because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part, because the antigens utilized were mainly monomers that did not display quaternary structure epitopes found on E-dimers and higher order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E-homodimers and DENV2 E-monomers. The stabilized DENV2 homodimers but not monomers were efficiently recognized by virus specific and flavivirus cross-reactive potent neutralizing antibodies that have been mapped to quaternary structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold higher levels of virus specific neutralizing IgG that recognized epitopes that were different from the epitopes recognized by lower level neutralizing antibodies induced by monomers. The dimer induced a stronger ED-I and ED-II targeted response, while the monomer antigens stimulated an ED-III epitope response and induced fusion loop epitope antibodies that are known to facilitate ADE. This study shows DENV E subunit antigens that have been designed to mimic the structural organization of viral surface are better vaccine antigens than E protein monomers.

    IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against 4 different dengue stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations, but have historically performed poorly as vaccine antigens, because the previously tested versions were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV virus particle surfaces when designing subunit vaccines.

  • Isolation and characterization of the first freshwater cyanophage infecting Pseudanabaena [Genetic Diversity and Evolution]

  • Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage PA-SR01 which was isolated from Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tail-less morphology. It is a double-stranded DNA virus with a 137,012 bp genome. Functional annotation for the predicted open reading frames (ORFs) of PA-SR01 genome identified genes with putative function related to DNA metabolism, structural protein, lysis, host-derived metabolic genes and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that Phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles.

    IMPORTANCE This study presents the isolation of the very first freshwater cyanophage PA-SR01 infecting Pseudanabaena and fills important knowledge gap on freshwater cyanophage as well as cyanophages infecting Pseudanabaena.

  • Axl deficiency promotes the neuroinvasion of Japanese encephalitis virus by enhancing IL-1{alpha} production from pyroptotic macrophages [Pathogenesis and Immunity]

  • Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis (JE), which has an unclear pathogenesis. Despite vaccination, thousands of deaths attributed to JE are reported annually. In this study, we report that mice deficient for Axl, a receptor tyrosine kinase that plays multiple roles in flaviviral infection, displayed greater mortality upon JEV infection. The effect of Axl deficiency on JEV infection was mediated by markedly elevated serum interleukin-1aalpha; (IL-1aalpha;) levels, which devastated the blood-brain-barrier and promoted viral neuroinvasion within 24 h post infection. Using an in situ infection model, we showed that dead macrophages were the primary source of observed increased serum IL-1aalpha; levels. Axl deficiency enhanced cell death and caused pyroptosis in 80% of JEV-infected macrophages by disrupting PI3K-Akt signaling. Intriguingly, the primary effector released by pyroptotic macrophages in our model was IL-1aalpha; rather than IL-1bbeta;. Finally, we assessed the effect of an IL-1aalpha; antagonist and demonstrated that it effectively prevented the incidence of JE. Our results indicate that Axl plays a protective role in JEV infection, identify IL-1aalpha; released by pyroptotic macrophages as a crucial factor promoting JEV neuroinvasion, and suggest that an IL-1aalpha; antagonist may be a candidate for JE therapy.

    IMPORTANCE Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese encephalitis (JE), the most commonly diagnosed viral encephalitis worldwide. The fatality rate of JE is 20%, and nearly half of the surviving patients develop neuropsychiatric sequelae. Axl is a receptor tyrosine kinase that plays multiple roles in flaviviral infections. Currently, the involvement of Axl in JEV infection remains enigmatic. In this study, we demonstrate that Axl impedes the pathogenesis of severe JE in mice by maintaining blood-brain-barrier (BBB) integrity and restricting viral neuroinvasion. Furthermore, serum IL-1aalpha; is a key mediator of this process and is primarily released by JEV-infected pyroptotic macrophages to elicit BBB breakdown, while an IL-1aalpha; antagonist can effectively reduce the incidence of severe JE. Our work uncovers the protective role of Axl in antagonizing severe JE and shows that the use of IL-1aalpha; antagonist may be a promising tactic to prevent severe JE.

  • A conformational escape reaction of HIV-1 against an allosteric integrase inhibitor [Vaccines and Antiviral Agents]

  • HIV-1 often acquires drug-resistant mutations in spite of the benefits of antiretroviral therapy (ART). HIV-1 integrase (IN) is essential for concerted integration of HIV-1 DNA into the host genome. IN further contributes to HIV-1 RNA binding, which is required for HIV-1 maturation. Non-catalytic site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which performs anti-HIV-1 activity by a multimodal mode of action such as inhibition of IN-LEDGF/p75 interaction in the early stage and disruption of functional IN multimerization in the late stage of HIV-1 replication. Here, we show that IN undergoes an adaptable conformational change to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated four amino acid (AA) mutations by passage 26 (P26) in the IN-encoding region. We employed HPLC, thermal stability assay, and X-ray crystallographic analysis to show that some AA mutations affect the stability and/or dimerization interface of the IN catalytic core domains (CCD), potentially resulting in severely decreased multimerization of full-length IN proteins (IN under-multimerization). This under-multimerized IN via the NCINI-related mutations was stabilized by HIV-1 RNA and restored to the same level as HIV-1 wild type in the viral particles. Recombinant HIV-1 clones with IN under-multimerization propagated similarly as HIV-1 wild type. Our study revealed that HIV-1 can eventually countervail NCINI-induced IN over-multimerization by IN under-multimerization as one of the escape mechanisms. Our findings provide information on the understanding of IN multimerization with or without HIV-1 RNA and may influence the development of anti-HIV-1 strategies.

    IMPORTANCE Understanding the mechanism of HIV-1 resistance to anti-HIV-1 drugs would lead to the development of novel drugs with increased efficiency, resulting in more effective ART. ART composed of more potent and long-acting anti-HIV-1 drugs can greatly improve drug adherence and also provide HIV-1 prevention such as pre-exposure prophylaxis. NCINIs with the multimodal mode of action exert potent anti-HIV-1 effects through IN over-multimerization during HIV-1 maturation. However, HIV-1 can acquire some mutations which cause IN under-multimerization to alleviate NCINI-induced IN over-multimerization. This under-multimerized IN was efficiently stabilized by HIV-1 RNA and restored to the same level as HIV-1 wild type. Our findings revealed that HIV-1 eventually acquires such conformational escape reaction to overcome the unique NCINI actions. The investigation into the drug-resistant mutations associated with HIV-1 protein multimerization may facilitate the elucidation of its molecular mechanism and functional multimerization, allowing us to develop more potent anti-HIV-1 drugs and unique treatment strategies.

  • Genetically and antigenically divergent influenza A(H9N2) viruses exhibit differential replication and transmission phenotypes in mammalian models [Pathogenesis and Immunity]

  • Low pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Co-circulation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.

    IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world, and for over twenty years, have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported and was found to exhibit properties of adaptation to humans and, importantly, show similarities to strains isolated from the live bird markets of Vietnam.

  • A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition [Structure and Assembly]

  • Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the pre-fusion conformers of llsquo;Class I' viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple ELISA-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of pre-fusion conformation at elevated temperatures, but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GPCL. Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.

    Importance The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

  • Interferon-induced Protein-44 and Interferon-induced Protein 44-like restrict replication of Respiratory Syncytial Virus [Pathogenesis and Immunity]

  • Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defence. We have previously used a bioinformatic screen to identify two interferon stimulated genes (ISG) with poorly characterised function, Interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in Respiratory Syncytial Virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model we investigated the antiviral capability of these genes in the control of RSV replication. Over-expression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time post infection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.

    IMPORTANCE RSV infects all children under two years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without pre-defined risk factors is in part mediated at the anti-viral gene level. But there is a large array of anti-viral genes, particularly in the ISG family about which the mechanism is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of over-expression and knockout studies we show that the genes are anti-viral and anti-proliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections and IFI44L can serve as a diagnostic bio-marker of viral infection.

  • Limited cross-protection provided by prior infection contributes to high prevalence of influenza D viruses in cattle [Pathogenesis and Immunity]

  • Since its detection in swine, Influenza D virus (IDV) has been shown to be present in multiple animal hosts, and bovines have been identified as its natural reservoir. However, it remains unclear how IDVs emerge, evolve, spread, and maintain in bovine populations. Through multiple years of virological and serological surveillance in a single order-buyer cattle facility in Mississippi, we showed consistently high seroprevalence of IDVs in cattle, and recovered a total of 32 IDV isolates from both healthy and sick animals including those with antibodies against IDV. Genomic analyses of these isolates along with those isolated from other areas showed that active genetic reassortment occurred in IDV and that five reassortants were identified in the Mississippian facility. Two antigenic groups were identified through antigenic cartography analyses for these 32 isolates and representative IDVs from other areas. Remarkably, existing antibodies could not protect cattle from experimental reinfection with IDV. Additional phenotypic analyses demonstrated variations in growth dynamics and pathogenesis in mice between viruses independent of genomic constellation. In summary, this study suggests that, in addition to epidemiological factors, the ineffectiveness of pre-existing immunity and cocirculation of a diverse viral genetic pool could facilitate its high prevalence in animal populations.

    IMPORTANCE Influenza D viruses (IDVs) are panzootic in multiple animal hosts, but the underlying mechanism is unclear. Through multiple years of surveillance in the same order-buyer cattle facility, 32 IDV isolates were recovered from both healthy and sick animals including those with evident antibodies against IDV. Active reassortment occurred in the cattle within this facility and in those across other areas, and multiple reassortants co-circulated in animals. These isolates are shown with a large extent of phenotypic diversity in replication efficiency and pathogenesis but little in antigenic properties. Animal experiment demonstrated that existing antibodies could not protect cattle from experimental reinfection with IDV. This study suggests that, in addition to epidemiological factors, limited protection from pre-existing immunity against IDVs in cattle herds and cocirculation of a diverse viral genetic pool likely facilitate the high prevalence of IDVs in animal populations.

  • SARS-CoV-2 vaccines: 'Warp Speed needs mind melds not warped minds [Minireviews]

  • In this review, we address issues that relate to the rapid "Warp Speed" development of vaccines to counter the COVID-19 pandemic. We review the antibody response that is triggered by SARS-CoV-2 infection of humans, and how it may inform vaccine research. The isolation and properties of neutralizing monoclonal antibodies from COVID-19 patients provide additional information on what vaccines should try to elicit. The nature and longevity of the antibody response to coronaviruses are relevant to the potency and duration of vaccine-induced immunity. We summarize the immunogenicity of leading vaccine candidates tested to date in animals and humans, and discuss the outcome and interpretation of virus-challenge experiments in animals. By far the most immunogenic vaccine candidates for antibody responses are recombinant proteins, which are not included in the initial wave of "Warp Speed" immunogens. A substantial concern for SARS-CoV-2 vaccines is adverse events, which we review by considering what was seen in studies of SARS-CoV-1 and MERS-CoV vaccines. We conclude by outlining the possible outcomes of the "Warp Speed" vaccine program, which range from the hoped-for rapid success to a catastrophic adverse influence on vaccine uptake generally.

  • Epstein Barr virus genomes reveal population structure and type 1 association with endemic Burkitt lymphoma [Genetic Diversity and Evolution]

  • Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is distinguished by its inclusion of Epstein-Barr virus (EBV). In order to better understand the impact of EBV variation in eBL tumorigenesis, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (N=58) and healthy controls (N=40) residing in the same geographic region in Kenya. Using our unbiased methods, we found that EBV type 1 was significantly more prevalent in eBL patients (74.5%) compared to healthy children (47.5%) (OR=3.24, 95% CI=1.36 - 7.71, P=0.007), as opposed to similar proportions in both groups. Controlling for EBV type, we also performed a genome-wide association study identifying 6 nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. Additionally, viruses isolated from plasma of eBL patients were identical to their tumor counterpart consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions as well as one novel genome with a 20 kb deletion resulting in the loss of multiple lytic and virion genes. Comparing EBV types, viral genes displayed differential variation rates as type 1 appeared to be more divergent while type 2 demonstrated novel substructures. Overall, our findings highlight the complexities of EBV population structure and provide new insight into viral variation, potentially deepening our understanding of eBL oncogenesis.

    Importance bbull; Improved viral enrichment methods conclusively demonstrate EBV type 1 as more prevalent in eBL patients compared to geographically matched healthy controls, which previously underrepresented the prevalence of EBV type 2.

    bbull; Genome-wide association analysis between cases and controls identifies 6 eBL-associated nonsynonymous variants in EBNA1, EBNA2, BcLF1, and BARF1 genes.

    bbull; Analysis of population structure reveals that EBV type 2 exists as two genomic sub groups, and was more commonly found in females than male eBL patients.

  • The BHLF1 Locus of Epstein-Barr Virus Contributes to Viral Latency and B-Cell Immortalization [Transformation and Oncogenesis]

  • The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform non-coding functions during virus replication, though an open reading frame is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression, and to be more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV post-transcriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long non-coding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle.

    IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. Establishment of latent infection, which is life long and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidation of how these EBV products contribute to latency is crucial to understanding EBV's role in specific malignancies, and ultimately to clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

  • Mitochondrial import of dengue virus NS3 protease and cleavage of GrpEL1, a co-chaperone of mtHsp70 [Virus-Cell Interactions]

  • Dengue virus infections pose a significant threat to human health at present, which is reported from nearly 140 countries. The genome of this virus encodes three structural and seven non-structural (NS) proteins along with two un-translated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis.Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions. But the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, dependent on its innate mitochondrial transport signal (MTS). Transient transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (1-464 amino acids of NS3) also targets the mitochondria. Intriguingly, reduced levels of the matrix localized GrpEL1, a co-chaperone of mtHsp70, were noticed in NS3pro, NS3pro-helicase expressing, and virus-infected cells. Upon using purified components, GrpEL1 undergoes cleavage, and the sites have been mapped to KR81A and QR 92S. Importantly, the levels of GrpEL1 are seriously compromised in severe dengue infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1 as a cleavage target, and thereby providing new avenues for the dengue research and the design of potential therapeutics.

    IMPORTANCE About 40% of the world's population is at the risk of dengue virus infections. There is no specific drug or potential vaccine for these infections until now. Lack of complete understanding about the pathogenesis is one of the hurdles for developing antivirals for this virus infection. In the present study, we show that the virus-coded protease imports to the mitochondrial matrix, which is the first-ever report with reference to the animal and human viruses. The analysis indicated that the observed mitochondrial import is due to the inherited mitochondrial transport signal. We also show that the matrix localized GrpEL1, a co-chaperone of mtHsp70, is also the substrate of dengue virus protease, as observed in in vitro, ex vivo, virus-infected cells, and dengue virus-infected clinical samples. Hence our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing anti-dengue therapeutics.

  • The measles virus V protein binding site to STAT2 overlaps with that of IRF9 [Structure and Assembly]

  • Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies, and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interaction between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is one order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size-exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N and C-terminal domains, from pre-formed complexes of STAT2-core/IRF-associated-domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt pre-assembled interferon-stimulated gene factor 3.

    IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase-signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferonnndash;mediated antiviral gene expression.

  • P108 and T109 on the E2 glycoprotein Domain I are critical for the adaptation of classical swine fever virus to rabbits but not the virulence in pigs [Virus-Cell Interactions]

  • The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs in contrast with the highly virulent CSFV Shimen strain. Previously, we have demonstrated that P108 and T109 on the E2 glycoprotein Domain I (E2P108-T109 on the E2DomainI) rather than R132, S133 and D191 on the Domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR). However, it remains elusive that whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring the E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that the E2P108-T109 or E2DomainI but not E2DomainII of C-strain render vSM-HCLVErns to be adaptive to rabbits, suggesting that the E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confer the Shimen strain ATR, creating new rabbit-adaptive CSFVs. Mechanistically, the E2P108-T109-Erns of C-strain mediate viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that the E2P108-T109-Erns of C-strain do not affect viral virulence compared with the Shimen strain. Conversely, the substitution of the E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and virulence in pigs are not overlapping. Our findings provide new insights into the adaptation mechanism of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.

    IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually lead to adaptation in cell cultures or non-susceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to non-susceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that the P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of rabbit-adaptive C-strain confer the adaptation of the highly virulent CSFV Shimen strain to rabbits by affecting viral entry during infection, but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular basis of CSFV adaptation to rabbits and attenuation in pigs.

  • Long noncoding RNA NKILA regulates HIV-1 replication and latency through repressing NF-ĸB signaling [Virus-Cell Interactions]

  • The lncRNA NKILA was recently identified as a negative regulator of NF-ĸB signaling and plays an important role in the development of various cancers. It is well known that NF-ĸB-mediated activation of human immunodeficiency virus-1 (HIV-1) LTR-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays an essential role in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-ĸB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms, as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assay revealed that NKILA expression abolishes the recruitment of p65 to the duplicated ĸB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-ĸB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA mainly due to reduced acetylation of Histone K27 on the promotor of NKILA by HIV-1 infection which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon PMA stimulation, while ectopic NKILA suppressed the reactivation in a well-established clinical model of withdrawal of azidothymidine (AZT) in vitro. These findings improve our understanding of the functional suppression of HIV-1 replication and latency by the lncRNA NKILA through NF-ĸB signaling.

    IMPORTANCE The NF-ĸB pathway plays a key role in HIV-1 replication and reactivation of HIV-1 latency. A regulator inhibiting NF-ĸB activation may be a promising therapeutic strategy against HIV-1. Recently, NF-ĸB interacting lncRNA (NKILA) was identified to suppress the development of different human cancers by inhibiting IKK-induced IĸB phosphorylation and NF-ĸB pathway activation, whereas the relationship between the lncRNA NKILA and HIV-1 replication is still unknown. Here, our results showed that the lncRNA NKILA inhibits HIV-1 replication and reactivation by suppressing HIV-1 LTR-driven transcription initiation. Moreover, NKILA inhibited the replication of HIV-1 clones with different coreceptor tropisms. This project may reveal a target for the development of novel anti-HIV drugs.

  • A conserved amino acid in the C-terminus of HPV E7 mediates binding to PTPN14 and repression of epithelial differentiation [Virus-Cell Interactions]

  • The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity.

    IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment we clearly demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte lifespan. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.

  • Role of Marek's disease virus encoded US3 serine/threonine protein kinase in regulating MDV Meq and cellular CREB phosphorylation [Virus-Cell Interactions]

  • Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the Alphaherpesvirinae subfamily. Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. Functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological function of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild type MDV US3, but not kinase dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of CREB responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase dead recombinant MDV, we identified US3 responsive MDV genes during infection, and found that the majority of US3 responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation-sequencing (ChIP-Seq) studies determined that some US3 regulated genes co-localized with Meq (MDV encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which are also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV infected chicken embryonic fibroblasts in a phosphorylation dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of CREB/Meq heterodimer, which targets cellular and viral gene expression.

    IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using MDV genome qRT-PCR array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step towards understanding of host-pathogen interaction through activation of signaling cascades.

  • CD300LF polymorphisms of inbred mouse strains confer resistance to murine norovirus infection in a cell type-dependent manner [Virus-Cell Interactions]

  • Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared MNV susceptibility of cells from different mouse strains and identified polymorphisms in murine CD300LF, which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains, which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4 amino acid difference at the CC' loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in non-permissive cells. Collectively, our data suggest the existence of a cell-type-specific modifier of MNV entry.

    IMPORTANCE MNV is a prevalent model system for studying human norovirusnndash;the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele that functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell-type specific modifiers of CD300LF-dependent MNV entry.

  • Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity [Virus-Cell Interactions]

  • Many RNA viruses encode a proof-reading deficient, low fidelity RNA dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart anti-viral therapies. 3Dpol, the RdRp of the Foot-and-Mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N-terminus encodes a nuclear localization signal (NLS) sequence, MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol. We identify two residues of 3Dpol NLS -T19 and L21- that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA and mutagen (RTP) bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network including D165 in motif F and R416 at the C-terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first 6 residues of the MRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.

    IMPORTANCE: In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol: T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs and displayed reduced replicative fitness when compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggests that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

  • hnRNP K is a novel ITAF that negatively regulates foot-and-mouth disease virus translation and replication and is antagonized by viral 3C protease [Virus-Cell Interactions]

  • Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region. Regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition is antagonized by the viral 3C protease through cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K1-364, retains partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and modulation of ITAFs, providing new insights into translational control during viral infection.

    IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1-364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K364-465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus, and provide new insights into translational control during viral infection.

  • Hazara nairovirus requires COPI components in both Arf1-dependent and Arf1-independent stages of its replication cycle [Virus-Cell Interactions]

  • Hazara nairovirus (HAZV) is an enveloped tri-segmented negative strand RNA virus classified within the Nairoviridae family of the Bunyavirales order, and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly and egress is poorly understood. Here, we generated a recombinant HAZV expressing eGFP and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi and ER as well as intra-Golgi transport. We showed the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle; an early stage prior to and including gene expression, and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titres by approximately 1000-fold. Treatment of HAZV-infected cells with brefeldin-A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed this late COPI-dependent stage was Arf1-dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1-independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a non-canonical Arf1-independent process suggests COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.

    IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analysed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle; an early stage prior to and including gene expression, and also a later stage during assembly of infectious virus, with COPI-knockdown reducing titres by approximately 1000-fold. Importantly, while the late stage was Arf1-dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1-independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions, and suggest a new Arf1-independent role for components of the COPI coatomer complex.

  • Human desmoglein-2 and human CD46 mediate HAdV55 infection but human desmoglein-2 plays the major roles [Virus-Cell Interactions]

  • Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. Here, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Non-permissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although non-transgenic mice were resistant to HAdV55 infection, infection of HAd55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics.

    IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immuno-competent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise non-transgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

  • TNPO3-mediated nuclear entry of the Rous sarcoma virus Gag protein is independent of the cargo-binding domain [Virus-Cell Interactions]

  • Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLS) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-aalpha;, which recruits importin-bbeta; to mediate nuclear entry. The second NLS, which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functionality of importin aalpha;/bbeta; and importin-11 has been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 mediates nuclear entry of Gag and directly binds to Gag. To our surprise, this interaction did not require the cargo-binding domain of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3 including SR-domain containing splicing factors and tRNAs that re-enter the nucleus. These results suggest that RSV hijacks the host nuclear import pathway using a unique mechanism, potentially allowing other cargo to bind TNPO3 simultaneously.

    IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the cargo binding domain of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

  • Hypervariable Domain of nsP3 of Eastern Equine Encephalitis Virus is a Critical Determinant of Viral Virulence. [Pathogenesis and Immunity]

  • Eastern equine encephalitis virus (EEEV) is the most pathogenic member of the Alphavirus genus in the Togaviridae family. This virus continues to circulate in the New World and has a potential for deliberate use as a bioweapon. Despite the public health threat, to date, no attenuated EEEV variants have been applied as live EEEV vaccines. Our previous studies demonstrated the critical function of the hypervariable domain (HVD) in EEEV nsP3 in the assembly of viral replication complexes (vRCs). EEEV HVD contains short linear motifs that recruit host proteins required for vRC formation and function. In this study, we developed a set of EEEV mutants, which contained combinations of deletions in nsP3 HVD and clustered mutations in capsid protein, and tested the effects of these modifications on EEEV infection in vivo. These mutations had cumulative negative effects on viral ability to induce meningoencephalitis. The deletions of two critical motifs, which interact with the members of cellular FXR and G3BP protein families, made EEEV no longer neurovirulent. The additional clustered mutations in capsid protein, which affect its ability to induce transcriptional shutoff, diminished EEEV's ability to develop viremia. Most notably, despite the inability to induce detectable disease, the designed EEEV mutants remained highly immunogenic and after a single dose, protected mice against subsequent infection with wt EEEV. Thus, alterations of interactions of EEEV HVD and likely HVDs of other alphaviruses with host factors represent an important direction of development of highly attenuated viruses that can be applied as live vaccines.

    IMPORTANCE Hypervariable domains (HVDs) of alphavirus nsP3 proteins recruit host proteins into viral replication complexes. The sets of HVD-binding host factors are specific for each alphavirus, and we have previously identified those specific for EEEV. The results of this study demonstrate that the deletions of the binding sites of G3BP and FXR protein families in nsP3 HVD of EEEV make virus avirulent for mice. Mutations in the nuclear localization signal in EEEV capsid protein have an additional negative effect on viral replication in vivo. Despite the inability to cause a detectable disease, the double HVD and triple HVD/capsid mutants induce high levels of neutralizing antibodies. Single immunization protects mice against the following infection with the highly pathogenic North American strain of EEEV. High safety, the inability to revert to wild type phenotype and high immunogenicity make the designed mutants attractive vaccine candidates for EEEV infection.

  • Meta-transcriptomic analysis of virus diversity in urban wild birds with paretic disease [Genetic Diversity and Evolution]

  • Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spill-over to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased meta-transcriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case the presence of the virus was confirmed by RT-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular and neuropathology in birds of the Corvidae and Artamidae families, and neuropathology in members of the Psittaculidae. The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock and human health.

    IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome, and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free ranging wildlife, and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

  • Reovirus core proteins {lambda}1 and {sigma}2 promote stability of disassembly intermediates and influence early replication events [Structure and Assembly]

  • The capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of mmu;1-3 heterohexamers which surround the core. The core is comprised of 1 decamers held in place by 2. After entry into the endosome, 3 is proteolytically degraded and mmu;1 is cleaved and exposed to form ISVPs. ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of mmu;1 peptides which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work has identified regions or specific residues within reovirus outer capsid proteins that impact the efficiency of cell entry. We examined the functions of the core proteins 1 and 2. We generated a reovirus T3D reassortant that carries strain T1L derived 2 and 1 proteins (T3D/T1L L3S2). This virus displays lower ISVP stability and therefore converts to ISVP*s more readily. To identify the molecular basis for lability of T3D/T1L L3S2, we screened for hyper-stable mutants of T3D/T1L L3S2 and identified three point mutations in mmu;1 that stabilize ISVPs. Two of these mutations are located in the C-terminal region of mmu;1, which has not previously been implicated in controlling ISVP stability. Independent from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells in comparison to T3D. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection.

    IMPORTANCE Protein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. In comparison to the parental T3D strain, we found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of the virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

  • IFN-{alpha} Induces Multiple Cellular Proteins that Coordinately Suppress Hepadnaviral cccDNA Transcription [Virus-Cell Interactions]

  • Covalently closed circular (ccc) DNA of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-aalpha;) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that down-regulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-aalpha; suppression of cccDNA transcription. On the contrary, ectopic expression of STAT1, SMCHD1 or PML significantly reduced cccDNA transcription activity. SMCHD1 is a non-canonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1 and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-aalpha;-induced post-translational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1 and PML may partly mediate the suppressive effect of IFN-aalpha; on hepadnaviral cccDNA transcription.

    IMPORTANCE Pegylated IFN-alpha is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-aalpha; in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-aalpha;. By a loss-of-function genetic screening of individual ISGs on hepadnaviral mRNAs transcribed from cccDNA, we found that down-regulating the expression of STAT1, SMCHD1 or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the post-translational modifications of cccDNA-associated histones similar to those induced by IFN-aalpha; treatment. We have thus identified three IFN-aalpha;-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-aalpha; silencing of hepadnaviral cccDNA transcription.

  • Zika Virus sfRNA Generation Requires Cooperativity between Duplicated RNA Structures that Are Essential for Productive Infection in Human Cells [Genome Replication and Regulation of Viral Gene Expression]

  • Zika virus (ZIKV) is an emerging flavivirus mainly transmitted by mosquitoes that represents a global health threat. A common feature of flavivirus infected cells is the accumulation of viral non-coding subgenomic RNAs by partial degradation of the viral genome, known as sfRNAs, involved in immune evasion and pathogenesis. Although a great effort is being placed to understand the mechanism by which these sfRNAs function during infection, the picture of how they work is still incomplete. In this study, we developed new genetic tools to dissect the functions of ZIKV RNA structures for viral replication and sfRNA production in mosquito and human hosts. ZIKV infections mostly accumulate two kinds of sfRNAs, sfRNA1 and sfRNA2, by stalling genome degradation upstream of duplicated stem loops (SLI and SLII) of the viral 3'UTR. Although the two SLs share conserved sequences and structures, different functions have been found for ZIKV replication in human and mosquito cells. While both SLs are enhancers for viral infection in human cells, they play opposite roles in the mosquito host. The dissection of determinants for sfRNA formation indicated a strong cooperativity between SLI and SLII, supporting a high order organization of this region of the 3'UTR. Using recombinant ZIKV with different SLI and SLII arrangements, which produce different types of sfRNAs or lack the ability to generate these molecules, revealed that at least one sfRNA was necessary for efficient infection and transmission in Aedes aegypti mosquitoes. Importantly, we demonstrate an absolute requirement of sfRNAs for ZIKV propagation in human cells. In this regard, viruses lacking sfRNAs, constructed by deletion of the region containing SLI and SLII, were able to infect human cells but the infection was rapidly cleared by antiviral responses. Our findings are unique for ZIKV since previous studies with other flaviviruses with deletions of analogous regions of the genome, including dengue and West Nile viruses, accumulated distinct species of sfRNAs and were infectious in human cells. We conclude that flaviviruses share common strategies for sfRNA generation, but they have evolved mechanisms to produce different kinds of these RNAs to accomplish virus-specific functions.

    IMPORTANCE Flaviviruses are important emerging and reemerging human pathogens. Understanding the molecular mechanisms for viral replication and evasion of host antiviral responses are relevant to develop control strategies. Flavivirus infections produce viral non-coding RNAs, known as sfRNAs, involved in viral replication and pathogenesis. Here, we dissected molecular determinants for Zika virus sfRNA generation in the two natural hosts, human cells and mosquitoes. We found that two RNA structures of the viral 3'UTR operate in a cooperative manner to produce two species of sfRNAs, and that the deletion of these elements have a profoundly different impact on viral replication in the two hosts. Generation of at least one sfRNA was necessary for efficient Zika virus infection of Aedes aegypti mosquitoes. Moreover, recombinant viruses with different 3'UTR arrangements revealed an essential role of sfRNAs for productive infection in human cells. In summary, we define molecular requirements for Zika virus sfRNA accumulation and provide new ideas of how flavivirus RNA structures have evolved to succeed in different hosts.

  • Induction of rod-shaped structures by herpes simplex virus glycoprotein I [Structure and Assembly]

  • The envelope glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used antibody mAb3104 and a newly-made peptide antibody to the gI extracellular domain (ECD) (aa.110-202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-ordered form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce rod-shaped structure was not required for viral replication and spread in cell culture, but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.

    IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here we report a novel property of this molecule, namely induction of rod-shaped structures, which appeared to represent a higher-ordered form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structure correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed lights on the biological properties of HSV-1 gI and may have important implications in understanding the viral pathogenesis.

  • Protein S-nitrosylation of Human Cytomegalovirus pp71 inhibits its ability to limit STING antiviral responses [Cellular Response to Infection]

  • Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that has co-evolved with its host and in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress induced, post-translational modification (PTM), protein S-nitrosylation, on viral proteins to determine the biological impact on viral replication.

    We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including STING activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread.

    IMPORTANCE In order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. Herein, we identified a previously unreported post-translational modification of pp71, protein S-nitrosylation. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71post-translational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this post-translational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.

  • Identification of common deletions in the spike protein of SARS-CoV-2 [Genetic Diversity and Evolution]

  • SARS-CoV-2 is a novel coronavirus first identified in December 2019. Notable features make SARS-CoV-2 distinct from most other previously-identified Betacoronaviruses, including the receptor binding domain of SARS-CoV-2 and a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells, however the deletion of QTQTN may restrict late phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and half of 24 in vitro isolated viruses, whilst the deletion of NSPRRAR was identified in 3 in vitro isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo, (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage, and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro. These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here.

    Important notes The spike protein determines the infectivity and host range of coronaviruses. SARS-CoV-2 has two unique features in its spike protein, the receptor binding domain and an insertion of twelve nucleotides at the S1/S2 boundary resulting a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN) and investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR and its flanking sites are not fixed in vitro, thus there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo. Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

  • An HIV vaccine targeting the V2 region of Envelope induces a highly durable polyfunctional Fc-mediated antibody response in rhesus macaques [Vaccines and Antiviral Agents]

  • The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of non-neutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen is highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only ~5x reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.

    Importance The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively non-neutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

  • Porcine epidemic diarrhea virus (PEDV) ORF3 protein is transported through the exocytic pathway [Virus-Cell Interactions]

  • Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV) the only gene at this location, ORF3, encodes a 224-residues membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking nor about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell, similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. Also the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, an YxxOOslash; motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.

    IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogues in other coronaviruses have been studied to different extents - indicating they share an ion channel property - little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. Here we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

  • A novel approach to display structural proteins of HCV quasispecies in patients reveals a key role of E2 HVR1 in viral evolution [Genetic Diversity and Evolution]

  • Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. Here we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins. HCV genome (JFH1 strain) lacking the structural proteins-coding sequence can be efficiently rescued by ectopic expression of Core-E1-E2-P7-NS2 (Core-NS2) or Core-E1-E2-P7 (Core-P7)in trans, leading to production of single-round infectious virions designated HCVS. JFH1-based HCVS can be also rescued by expressing Core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies in vivo. Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from a same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution in vivo.

    IMPORTANCE A cell-culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. Here we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins (HCVS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution in vivo. Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.

  • Inactivating Three Interferon Antagonists Attenuates Pathogenesis of an Enteric Coronavirus [Pathogenesis and Immunity]

  • Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts into humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs including respiratory and enteric systems, as exemplified by the newly emerged SARS-CoV-2. The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural proteins 1, 15, and 16 individually, or combined in one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses, as compared to wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 post-infection. The icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivating these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.

    Author summary Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploit a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our study reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15 and 16, results in a highly attenuated virus that does not cause diarrhea in animals, and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live-attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

  • Trehalose Inhibits Human Immunodeficiency Virus type-1 Infection in Primary Human Macrophages and CD4+ T lymphocytes through Two Distinct Mechanisms [Vaccines and Antiviral Agents]

  • Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a non-toxic, non-reducing disaccharide that induces autophagy through an MTOR independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2 and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5. Additionally, trehalose down-regulated the expression of CCR5 in T cells, and CD4 in both T cells and macrophages which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar, trehalose, at doses safely achieved in humans inhibits HIV through two mechanisms: 1) decreased entry through the down-regulation of CCR5 in T-cells, and decreased CD4 expression in both T-cells and macrophages; and 2) degradation of intracellular HIV through the induction of MTOR independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, non-toxic sugar.

    Importance: Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of MTOR has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV inhibitory effects of the MTOR independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T-cells through the down-regulation of CCR5 in T cells, and CD4 in both T cells and macrophages. Thus, we have shown that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

  • Vesicular stomatitis virus and DNA vaccines expressing Zika virus nonstructural protein 1 (NS1) induce substantial but not sterilizing protection against Zika virus infection [Vaccines and Antiviral Agents]

  • The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1-/- mice, neither NS1-based vaccine provided protection against a lethal high dose (105 PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (103 PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development.

    Importance Most Zika virus (ZIKV) vaccine research has focused on the E or prM-E proteins and the induction of high levels of neutralizing antibodies. However, these ZIKV neutralizing antibodies cross react with other flaviviruses, which may aggravate the disease via an Antibody Dependent Enhancement (ADE) mechanism. ZIKV NS1 protein may be an alternative antigen for vaccine development, as antibodies to NS1 do not bind to the virion, thereby eliminating the risk of ADE. Here we show that recombinant VSV and DNA vaccines expressing NS1, alone, confer partial protection against ZIKV infection in both immunocompetent and immunodeficient mice, highlighting the value of NS1 as a potential vaccine candidate.

  • Fusion of large polypeptides to human adenovirus type 5 capsid protein IX can compromise virion stability and DNA packaging capacity [Gene Delivery]

  • The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known if such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wildtype sized genomes. However, similar virions containing wildtype sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function.

    IMPORTANCE Human adenovirus (HAdV) capsid protein IX (pIX) is involved in stabilizing the virion, but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.

  • A genome-wide CRISPR-Cas9 screen reveals the requirement of host cell sulfation for Schmallenberg virus infection [Virus-Cell Interactions]

  • Schmallenberg virus (SBV) is an insect-transmitted Orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using a genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by solute carrier family 35 member B2 (SLC35B2). SBV cell surface attachment and entry were largely reduced upon knockout of SLC35B2, whereas reconstitution of SLC35B2 in these cells fully restores their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role for cell attachment and entry. Although to varying degrees, heparan sulfate was also found to be important to initiate infection by two other Bunyaviruses, La Crosse and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for efficient replication of SBV and other Bunyaviruses.

    IMPORTANCE SBV is a newly emerging Orthobunyavirus (family Bunyaviridae) that has spread rapidly across Europe since 2011 resulting in substantial economic losses in livestock farming. In this study, we performed an unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting their importance for host cell infection of Bunyaviruses. Thus, the present work provides crucial insights in virus-host interactions of important animal and human pathogens.

  • Porcine Reproductive and Respiratory Syndrome Virus Utilizes Viral Apoptotic Mimicry as an Alternative Pathway to Infect Host Cells [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), has led to enormous economic losses in global swine industry. Infection by PRRSV is previously shown to be via low pH-dependent clathrin-mediated endocytosis (CME) and CD163 functions as an essential receptor during viral infection. Despite much research focusing on it, PRRSV infection remains to be fully elucidated. In this study, we demonstrated that PRRSV externalized phosphatidylserine (PS) on the envelope as viral apoptotic mimicry, and infected host cells through T-cell immunoglobulin and mucin domain (TIM)-induced and CD163-involved macropinocytosis as an alternative pathway. In detail, we identified that PS receptor (PSR) TIM-1/4 recognized and interacted with PRRSV as viral apoptotic mimicry and subsequently induced macropinocytosis by the downstream Rho GTPases Rac1, cell division control protein 42 (Cdc42) and p21-activated kinase 1 (Pak1). Altogether these results expand our knowledge of PRRSV infection, which will support implications for prevention and control of PRRS.

    IMPORTANCE PRRS has caused huge economic losses to pig farming worldwide. Its causative agent, PRRSV, infects host cells through low pH-dependent CME and CD163 is indispensable during the process. Whether there exist alternative infection pathways for PRRSV arouses our interest. Here, we found that PRRSV exposed PS on its envelope and disguised as apoptotic debris. The PSR TIM-1/4 recognized PRRSV and induced the downstream signaling pathway to mediate viral infection via CD163-dependent macropinocytosis. The current work deepens our understanding of PRRSV infection, and provides clues for the development of drugs and vaccines against the virus.

  • Shedding-resistant HIV-1 envelope glycoproteins adopt downstream conformations that remain responsive to conformation-preferring ligands [Virus-Cell Interactions]

  • The HIV-1 envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4+ cells. Single-molecule Fluorescence Resonance Energy Transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pre-triggered conformation (State 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (State 2) into the three-CD4-bound open conformation (State 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream States 2 and 3. The structure of State 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pre-triggered State 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S), or by introducing a disulfide bridge between gp120 and gp41 designated "SOS" (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream towards States 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in State 1 by specific ligands such as the BMS entry inhibitors. The here-described identification of State 1-stabilizing conditions may enable structural characterization of the State 1 conformation of HIV-1 Env.

    IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pre-triggered (State 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pre-triggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in State 1, potentially facilitating structural characterization of this unknown conformational state.

  • Strict assembly restriction of peptides from rabbit hemorrhagic disease virus presented by rabbit MHC class I molecule RLA-A1 [Structure and Assembly]

  • Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Herein, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and P positions. Meanwhile, the rabbit molecule is featured with an uncommon residue combination of Gly53, Val55 and Glu56, making the 310 helix and the loop between the 310 and aalpha;1 helices closer to the aalpha;2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge on T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.

    IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of Rabbit haemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2 and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to human HLA-A*0201. In addition, uncommon residues Gly53, Val55 and Glu56 of RLA-A1 are located between 310 helix and aalpha;1 helix which makes the steric position of 310 helix in RLA-A1 much closer to the aalpha;2 helix than other mammalian MHC class I molecules. This special conformation between 310 helix and aalpha;1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.

  • Mutagenic analysis of Hazara nairovirus non-translated regions during single and multi-step growth identifies both attenuating and functionally-critical sequences for virus replication [Genome Replication and Regulation of Viral Gene Expression]

  • Hazara nairovirus (HAZV) is a member of the Nairoviridae family within the Bunyavirales order, and closely-related to Crimean-Congo hemorrhagic fever virus that is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative sense RNA named S, M and L, with non-translated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis, and by analogy with other segmented negative sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3' and 5' NTRs exhibit extensive terminal complementarity; the first eleven nucleotides are strictly conserved and form promoter element (PE) 1, with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multi-step growth, which revealed the 3' NTR was highly tolerant to change whereas several deletions of centrally-located nucleotides within the 5' NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth, and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest its signalling ability relies solely on inter-terminal base pairing, and is an independent cis-acting signalling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed.

    IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3' and 5' ends of nairoviral genes, called non-translated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle such as mRNA transcription and RNA replication, with other roles likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3' and 5' NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

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