|JVI Current Issue|
The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cellrrsquo;s innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2aalpha;. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR.
IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.
Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo, which were subsequently used to design a KdNS5B451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo. The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal.
IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.
Autosomal dominant STAT1 mutations in humans have been associated with chronic mucocutaneous candidiasis (CMC), as well as with increased susceptibility to herpesvirus infections. Prior studies have focused on mucosal and Th17-mediated immunity against Candida, but mechanisms of impaired antiviral immunity have not previously been examined. To begin to explore the mechanisms of STAT1-associated immunodeficiency against herpesviruses, we generated heterozygous STAT1 R274W knock-in mice that have a frequently reported STAT1 mutation associated in humans with susceptibility to herpesvirus infections. In primary macrophages and fibroblasts, we found that STAT1 R274W had no appreciable effect on cell-intrinsic immunity against herpes simplex virus 1 (HSV-1) or gammaherpesvirus 68 (HV68) infection. However, intraperitoneal inoculation of mice with HV68 was associated with impaired control of infection at day 14 in STAT1 R274W mice compared with that in wild-type (WT) littermate control animals. Infection of STAT1 R274W mice was associated with paradoxically decreased expression of IFN-stimulated genes (ISGs) and gamma interferon (IFN-), likely secondary to defective CD4+ and CD8+ T cell responses, including diminished numbers of antigen-specific CD8+ T cells. Viral pathogenesis studies in WT and STAT1 R274W mixed bone marrow chimeric mice revealed that the presence of WT leukocytes was sufficient to limit infection and that antigen-specific STAT1 R274W CD8+ T cell responses were impaired even in the presence of WT leukocytes. Thus, in addition to regulating Th17 responses against Candida, a STAT1 gain-of-function mutant impedes antigen-specific T cell responses against a common gammaherpesvirus in mice.
IMPORTANCE Mechanisms of immunodeficiency related to STAT1 gain of function have not been previously studied in an animal model of viral pathogenesis. Using virological and immunological techniques, we examined the immune response to HV68 in heterozygous mice that have an autosomal dominant mutation in the STAT1 coiled-coil domain (STAT1 R274W). We observed impaired control of infection, which was associated with diminished production of gamma interferon (IFN-), fewer effector CD4+ and CD8+ T cells, and a reduction in the number of antigen-specific CD8+ T cells. These findings indicate that a STAT1 gain-of-function mutation limits production of antiviral T cells, likely contributing to immunodeficiency against herpesviruses.
Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV; however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs, termed MPV196, MPV201, MPV314, and MPV364, are described. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro. Binding studies with monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity for monomeric hMPV F compared to the other three mAbs, yet binding experiments with trimeric hMPV F showed limited differences in binding affinity, suggesting that MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, both of which cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests that it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.
IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope yet is unique from previously discovered site III mAbs, as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo and found that it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development and provide insights into hMPV vaccine development.
Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation.
IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.
Increased frequencies of immunosuppressive regulatory T cells (Tregs) are associated with gut lymphoid tissue fibrosis and dysfunction which, in turn, contribute to disease progression in chronic simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection. Mesenteric lymph nodes (MLNs), which drain the large and small intestine, are critical sites for the induction and maintenance of gut mucosal immunity. However, the dynamics of Tregs in MLNs are not well understood due to the lack of accessibility to these tissues in HIV-infected individuals. Here, the dynamics of Tregs in blood and MLNs were assessed in SIV-infected rhesus macaques (RMs) following early antiretroviral drug (ARV) initiation. Early ARV initiation reduced T-cell immune activation, as assessed by HLA-DR/CD39 expression, and prevented the depletion of memory CCR6+ Th17 cells in both blood and MLNs. Untreated animals showed higher frequencies of Tregs, CD39+ Tregs, thymic Tregs, and new memory CD4 populations sharing similarity with Tregs as CTLA4+ PD1nndash; and CTLA4+ PD1nndash; FoxP3+ T cells. Despite early ARV treatment, the frequencies of these Treg subsets remained unchanged within the MLNs and, in contrast to blood normalization, the Th17/Treg ratio remained distorted in MLNs. Furthermore, our results highlighted that the expressions of IDO-1, TGFbbeta;1 and collagen-1 mRNA remained unchanged in MLN of ARV-treated RMs. ARV interruption did not affect T-cell immune activation and Th17/Treg ratios in MLN. Altogether, our data demonstrated that early ARV initiation within the first few days of SIV infection is unable to reduce the frequencies and homing of various subsets of Tregs within the MLNs which, in turn, may result in tissue fibrosis, impairment in MLN function, and HIV persistence.
IMPORTANCE Tregs contribute to SIV/HIV disease progression by inhibition of antiviral specific responses and effector T-cell proliferation. Tregs also cause tissue fibrosis via transforming growth factor bbeta;1 production and collagen deposition, which are associated with microbial translocation and generalized immune activation. Early ARV initiation upon viral exposure is recommended globally and results in improved immune function recovery and reduced viral persistence. Here, using an acute SIV infection model of rhesus macaques, we demonstrated for the first time that despite clear improvements in mucosal CD4 T cells, in contrast to blood, Treg frequencies in MLNs remained elevated following early ARV initiation. The particular Th17/Treg balance observed in MLNs can contribute, in part, to the maintenance of mucosal fibrosis during suppressive ARV treatment. Our results provide a better understanding of gut mucosal immune dynamics following early ARV initiation. These findings suggest that Treg-based treatments could serve as a novel immunotherapeutic approach to decrease gut mucosal damage during SIV/HIV infections.
Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus.
IMPORTANCE ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCKrrsquo;s antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus.
Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.
IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.
Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.
IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.
Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5' untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps.
IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.
CD69 is highly expressed on the leukocyte surface upon viral infection, and its regulatory role in the vaccinia virus (VACV) immune response has been recently demonstrated using CD69nndash;/nndash; mice. Here, we show augmented control of VACV infection using the anti-human CD69 monoclonal antibody (MAb) 2.8 as both preventive and therapeutic treatment for mice expressing human CD69. This control was related to increased natural killer (NK) cell reactivity and increased numbers of cytokine-producing T and NK cells in the periphery. Moreover, similarly increased immunity and protection against VACV were reproduced over both long and short periods in anti-mouse CD69 MAb 2.2-treated immunocompetent wild-type (WT) mice and immunodeficient Rag2nndash;/nndash; CD69+/+ mice. This result was not due to synergy between infection and anti-CD69 treatment since, in the absence of infection, anti-human CD69 targeting induced immune activation, which was characterized by mobilization, proliferation, and enhanced survival of immune cells as well as marked production of several innate proinflammatory cytokines by immune cells. Additionally, we showed that the rapid leukocyte effect induced by anti-CD69 MAb treatment was dependent on mTOR signaling. These properties suggest the potential of CD69-targeted therapy as an antiviral adjuvant to prevent derived infections.
IMPORTANCE In this study, we demonstrate the influence of human and mouse anti-CD69 therapies on the immune response to VACV infection. We report that targeting CD69 increases the leukocyte numbers in the secondary lymphoid organs during infection and improves the capacity to clear the viral infection. Targeting CD69 increases the numbers of gamma interferon (IFN-)- and tumor necrosis factor alpha (TNF-aalpha;)-producing NK and T cells. In mice expressing human CD69, treatment with an anti-CD69 MAb produces increases in cytokine production, survival, and proliferation mediated in part by mTOR signaling. These results, together with the fact that we have mainly worked with a human-CD69 transgenic model, reveal CD69 as a treatment target to enhance vaccine protectiveness.
Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO. In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO. We found that NOD/SCID/Cnndash;/nndash; c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo. HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.
IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.
The potential avian influenza pandemic remains a threat to public health, as the avian-origin influenza A(H7N9) virus has caused more than 1,560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low-pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity, as determined by a hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs.
IMPORTANCE The circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by a large number of human infections and the emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low-pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. The in vitro and in vivo experiments demonstrated comparable antigenicity of the G218E CVVs with that of their wild-type (WT) counterparts, and both the WT and the G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit preparedness against the threat of an A(H7N9) influenza pandemic.
Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.
IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.
Human T-cell leukemia virus type 1 (HTLV-1) causes multiple pathological effects, ranging from a form of leukemia to a spectrum of inflammation-mediated diseases. These diseases arise from one or several infected CD4+ T cells among thousands acquiring proliferation and survival advantages and ultimately becoming pathogenic. Given the low incidence of HTLV-1-associated diseases among carriers, such cellular evolutionary processes appear to occur rarely. Therefore, infectious spread of HTLV-1 within the T-cell population may be one underlying factor influencing disease development. Free HTLV-1 virions are poorly infectious, so infection of T cells relies on direct contact between infected and target cells. Following contact, virions pass to target cells through a virological synapse or cellular conduits or are transferred to target cells within an extracellular matrix. Lymphocyte functioning antigen 1 (LFA-1) on the surface of the target cell engaging with its ligand, ICAM-1, on the surface of the infected cell (effector cell) initiates and stabilizes cell-cell contact for infection. We found that stable expression of an HTLV-1 accessory protein, HTLV-1 bZIP factor (HBZ), in Jurkat T cells increases homotypic aggregation. This phenotype was attributed to elevated ICAM-1 expression in the presence of HBZ. Using a single-cycle replication-dependent luciferase assay, we found that HBZ expression in Jurkat cells (used as effector cells) increases HTLV-1 infection. Despite this effect, HBZ could not replace the critical infection-related functions of the HTLV-1 regulatory protein Tax. However, in HTLV-1-infected T cells, knockdown of HBZ expression did lead to a decrease in infection efficiency. These overall results suggest that HBZ contributes to HTLV-1 infectivity.
IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) causes a variety of diseases, ranging from a fatal form of leukemia to immune-mediated inflammatory diseases. These diseases occur rarely, arising from one or a small subset of virally infected cells infrequently evolving into a pathogenic state. Thus, the process of HTLV-1 cell-to-cell transmission within the host helps influence the probability of disease development. HTLV-1 primarily infects T cells and initially spreads within this cell population when virally infected T cells dock to uninfected target T cells and then transfer HTLV-1 virus particles to the target cells. Here we found that the viral protein HTLV-1 bZIP factor (HBZ) promotes infectivity. HBZ accomplishes this task by increasing the surface abundance of a cellular adhesion protein known as intercellular adhesion molecule 1 (ICAM-1), which helps initiate and stabilize contact (docking) between infected and target T cells. These results define a novel and unexpected function of HBZ, diverging from its defined functions in cellular survival and proliferation.
The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at aalpha;2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the aalpha;2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.
IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.
A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Ms) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally "stitching" together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Ms, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/M, and a Neu.
IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for aalpha;-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Ms and Neus, using electron microscopy. The finding of a M/M harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Ms are involved in the pathogenesis of IAV primary pneumonia.
One large open reading frame (ORF) encodes 10 potyviral proteins. We compared the accumulation of cylindrical inclusion (CI) protein from the middle, coat protein (CP) from the 3'end, and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5' RLUC was expressed from an rluc gene inserted between the P1 and helper component proteinase (HCPro) cistrons, and 3' RLUC was expressed from the gene inserted between the RNA polymerase and CP cistrons. Viral protein and RNA accumulation were quantitated (i) when expressed from PVA RNA in the presence of ectopically expressed genome-linked viral protein (VPg) and auxiliary proteins and (ii) at different time points during natural infection. The rate and timing of 3' RLUC and CP accumulation were found to be different from those of 5' RLUC and CI. Ectopic expression of VPg boosted PVA RNA, 3' RLUC, and, together with HCPro, CP accumulation, whereas 5' RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3' RLUC accelerated toward the end of infection. 5' RLUC accumulation, which was already pronounced at 2 days postinfection, increased moderately and stabilized to a constant level by day 5, whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism of how this occurs.
IMPORTANCE The results of this study suggest that the dynamics of potyviral protein accumulation are regulated differentially from the 3' end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.
Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems.
IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro. These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.
A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.
IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.
As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (aalpha;-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of aalpha;-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an aalpha;-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.
IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce aalpha;-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
Transforming growth factor bbeta; (TGF-bbeta;) has been shown to play a role in immunity against different pathogens in vitro and against parasites in vivo. However, its role in viral infections in vivo is incompletely understood. Using a neonatal mouse model of heterologous rhesus rotavirus (RV) vaccination, we show that the vaccine induced rotavirus-specific CD4 T cells, the majority of which lacked expression of KLRG1 or CD127, and a few regulatory rotavirus-specific CD4 T cells that expressed surface latency-associated peptide (LAP)nndash;TGF-bbeta;. In these mice, inhibiting TGF-bbeta;, with both a neutralizing antibody and an inhibitor of TGF-bbeta; receptor signaling (activin receptor-like kinase 5 inhibitor [ALK5i]), did not change the development or intensity of the mild diarrhea induced by the vaccine, the rotavirus-specific T cell response, or protection against a subsequent challenge with a murine EC-rotavirus. However, mice treated with anti-LAP antibodies had improved protection after a homologous EC-rotavirus challenge, compared with control rhesus rotavirus-immunized mice. Thus, oral vaccination with a heterologous rotavirus stimulates regulatory RV-specific CD4 LAP-positive (LAP+) T cells, and depletion of LAP+ cells increases vaccine-induced protection.
IMPORTANCE Despite the introduction of several live attenuated animal and human rotaviruses as efficient oral vaccines, rotaviruses continue to be the leading etiological agent for diarrhea mortality among children under 5 years of age worldwide. Improvement of these vaccines has been partially delayed because immunity to rotaviruses is incompletely understood. In the intestine (where rotavirus replicates), regulatory T cells that express latency-associated peptide (LAP) play a prominent role, which has been explored for many diseases but not specifically for infectious agents. In this paper, we show that neonatal mice given a live oral rotavirus vaccine develop rotavirus-specific LAP+ T cells and that depletion of these cells improves the efficiency of the vaccine. These findings may prove useful for the design of strategies to improve rotavirus vaccines.
Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the Caliciviridae family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 AAring; in physiological buffers. Since this unusual feature is shared among, and unique to, the Caliciviridae, it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (~3 AAring;) and the receptor CD300lf (~8 AAring;). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity.
IMPORTANCE Mouse norovirus and several other members of the Caliciviridae have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90ddeg; rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.
Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively.
IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.
Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae, is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV.
IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen.
Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theilerrrsquo;s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2aalpha; phosphorylation and, thus, acts downstream of eIF2aalpha;; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2aalpha; phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(Immiddot;C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA.
IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theilerrrsquo;s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.
Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.
IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.
The adenovirus (Ad) E4orf4 protein was reported to contribute to inhibition of ATM- and ATR-regulated DNA damage signaling during Ad infection and following treatment with DNA-damaging drugs. Inhibition of these pathways improved Ad replication, and when expressed alone, E4orf4 sensitized transformed cells to drug-induced toxicity. However, the mechanisms utilized were not identified. Here, we show that E4orf4 associates with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1) and that the association requires PARP activity. During Ad infection, PARP is activated, but its activity is not required for recruitment of either E4orf4 or PARP-1 to virus replication centers, suggesting that their association occurs following recruitment. Inhibition of PARP-1 assists E4orf4 in reducing DNA damage signaling during infection, and E4orf4 attenuates virus- and DNA damage-induced parylation. Furthermore, E4orf4 reduces PARP-1 phosphorylation on serine residues, which likely contributes to PARP-1 inhibition as phosphorylation of this enzyme was reported to enhance its activity. PARP-1 inhibition is important to Ad infection since treatment with a PARP inhibitor enhances replication efficiency. When E4orf4 is expressed alone, it associates with poly(ADP-ribose) (PAR) chains and is recruited to DNA damage sites in a PARP-1-dependent manner. This recruitment is required for inhibition of drug-induced ATR signaling by E4orf4 and for E4orf4-induced cancer cell death. Thus, the results presented here demonstrate a novel mechanism by which E4orf4 targets and inhibits DNA damage signaling through an association with PARP-1 for the benefit of the virus and impacting E4orf4-induced cancer cell death.
IMPORTANCE Replication intermediates and ends of viral DNA genomes can be recognized by the cellular DNA damage response (DDR) network as DNA damage whose repair may lead to inhibition of virus replication. Therefore, many viruses evolved mechanisms to inhibit the DDR network. We have previously shown that the adenovirus (Ad) E4orf4 protein inhibits DDR signaling, but the mechanisms were not identified. Here, we describe an association of E4orf4 with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1). E4orf4 reduces phosphorylation of this enzyme and inhibits its activity. PARP-1 inhibition assists E4orf4 in reducing Ad-induced DDR signaling and improves the efficiency of virus replication. Furthermore, the ability of E4orf4, when expressed alone, to accumulate at DNA damage sites and to kill cancer cells is attenuated by chemical inhibition of PARP-1. Our results indicate that the E4orf4nndash;PARP-1 interaction has an important role in Ad replication and in promotion of E4orf4-induced cancer-selective cell death.
|JVI Accepts: Articles Published Ahead of Print|
In spite of several decades of research focused on understanding the human herpes simplex virus 1 (HSV-1) biology, no tool has been developed to study its genome in a high throughput fashion. Here, we describe the creation of a transposon insertion mutant library of the HSV-1 genome. Using this tool, we aimed to identify novel viral regulators of type I interferon. HSV-1 evades the host immune system by encoding viral proteins that inhibit the type I interferon response. Applying differential selective pressure, we identified the three strongest viral IFN-I regulators in HSV-1. We report that the viral polymerase processivity factor UL42 interacts with the host transcription factor IRF-3, inhibiting its phosphorylation and downstream interferon-bbeta; gene transcription. This study represents a proof of concept for the use of high-throughput screening of the HSV-1 genome in investigating viral biology and offers new targets both for antiviral therapy and for oncolytic vector design.
IMPORTANCE This work is the first to report the use of a high-throughput mutagenesis method to study the genome of HSV-1. We report three novel viral proteins potentially involved in regulating the host type I interferon response. We describe a novel mechanism by which the viral protein UL42 is able to suppress production of interferon-bbeta;. The tool we introduce in this study can be used to study the HSV-1 genome in great detail to better understand viral genes functions.
Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene Ontology (GO) analysis was performed to functionally annotate these proteins, and the oligosaccharyltransferase (OST) complex was highly enriched. Functional studies conducted by using CRISPR-Cas9-mediated knockouts showed that STT3A and STT3B, the two catalytically active isoforms of the OST complex, are essential for the propagation of the recombinant arenavirus rLCMV/LASV GPC, mainly via affecting virus infectivity. Knockout of STT3B, but not STT3A, caused hypoglycosylation of LASV GP, indicating a preferential requirement of LASV for the STT3B-OST isoform. Furthermore, double knockout of magnesium transporter 1 (MAGT1) and tumor suppressor candidate 3 (TUSC3), two specific subunits of STT3B-OST, also caused hypoglycosylation of LASV GP and affected virus propagation. Site-directed mutagenesis analysis revealed that the oxidoreductase CXXC active site motif of MAGT1 or TUSC3 is essential for the glycosylation of LASV GP. NGI-1, a small-molecule OST inhibitor, can effectively reduce virus infectivity without affecting cell viability. The STT3B-dependent N-glycosylation of GP is conserved among other arenaviruses, including both the Old World (OW) and New World (NW) groups. Our study provided a systematic view of LASV GP-host interactions and revealed the preferential requirement of STT3B for LASV GP N-glycosylation.
IMPORTANCE Glycoproteins play vital roles in the arenavirus life cycle by facilitating virus entry and participating in the virus budding process. N-glycosylation of GPs is responsible for their proper functioning; however, little is known about the host factors on which the virus depends for this process. In this study, a comprehensive LASV GP interactome was characterized, and further study revealed that STT3B-dependent N-glycosylation was preferentially required by arenavirus GPs and critical for virus infectivity. The two specific thioredoxin subunits of STT3B-OST MAGT1 and TUSC3 were found to be essential for the N-glycosylation of viral GP. NGI-1, a small-molecule inhibitor of OST, also showed a robust inhibitory effect on arenavirus. Our study provides new insights into LASV GP-host interactions and extends the potential targets for the development of novel therapeutics against Lassa fever in the future.
Breast cancer is the second-leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian orthoreovirus (reovirus) selectively infects and kills transformed cells and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modulating the activity of host molecules and pathways. Here, we generated reassortant reoviruses by forward genetics with enhanced infective and cytotoxic properties in triple-negative breast cancer cells. From a high-throughput screen of small molecule inhibitors, we identified topoisomerase inhibitors as a class of drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of triple-negative breast cancer cells with topoisomerase inhibitors activates DNA damage response pathways and reovirus infection induces robust production of Type III, but not Type I, interferon. Although Type I and Type III IFN can activate STAT1 and STAT2, triple-negative breast cancer cellular proliferation is only negatively affected by Type I IFN. Together, these data show that reassortant viruses with a novel genetic composition generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple-negative breast cancer cells.
IMPORTANCE Patients afflicted by triple-negative breast cancer have decreased survival and limited therapeutic options. Reovirus infection results in cell death of a variety of cancers, but it is unknown if different reovirus types lead to triple-negative breast cancer cell death. In this study, we generated two novel reoviruses that more efficiently infect and kill triple-negative breast cancer cells. We show that infection in the presence of DNA-damaging agents enhances infection and triple-negative breast cancer cell killing by reovirus. These data suggest that a combination of a genetically engineered oncolytic reovirus and topoisomerase inhibitors may provide a potent therapeutic option for patients afflicted with triple-negative breast cancer.
Equine herpesvirus type 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the IFN-bbeta; response in vitro in equine endothelial cells (EECs) at 12 h post-infection (hpi). In the current study, we explored the molecular correlates of this inhibition as clues towards an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both TLR3 and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IRF7 mRNA only at 12 hpi. EHV-1 did not alter the cellular level of JAK1 at any time point. However, EHV-1 reduced the cellular level of expression of TYK2 at 12 hpi. Downstream of the JAK1-TYK2 signaling EHV-1 blocked the phosphorylation and activation of STAT2 when co-incubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type-I IFN sensitization and induction.
IMPORTANCE To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by EHV-1, especially the neurologic form. The interferon (IFN) system, of which type-I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type-I IFN system has been exploited successfully to treat other viral infections such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type-I IFN has indicated transient induction of type-I IFN production followed by a rapid shut-down in vitro in EECs. The significance of our study is the identification of certain steps in the type-I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1.
Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, like poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. Quantity and composition of bacterial communities were altered by treatment with the five antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis, while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 is more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ in their reliance on microbiota.
IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ in their reliance on microbiota.
We previously reported that HSV glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. Binding of gK to SPP is required for HSV-1 infectivity in vitro. SPP is a member of the -secretase family and mice lacking SPP are embryonic lethal. To determine how SPP affects HSV-1 infectivity in vivo, the SPP gene was deleted using a tamoxifen-inducible Cre recombinase driven by the ubiquitously expressed ROSA26 promoter. SPP mRNA was reduced by more than 93% in the cornea and trigeminal ganglia and 99% in the liver of tamoxifen injected mice, while SPP protein expression was reduced by 90% compared with control mice. Mice lacking SPP had significantly less HSV-1 replication in their eye as well as reduced gK, UL20, ICP0 and gB transcripts in their cornea and trigeminal ganglia (TG) compared with control mice. In addition, reduced infiltration of CD45+, CD4+, CD8+, F4/80+, CD11c+ and NK1.1+ T cells was observed in the cornea and TG of SPP-inducible knockout mice than in control mice. Finally, in the absence of SPP, latency was significantly reduced in SPP-inducible knockout mice compared to control mice. Thus, in this study we have generated SPP-inducible knockout mice and shown that the absence of SPP affects virus replication in the eye of ocularly infected mice and that this reduction is correlated with the interaction of gK and SPP. These results suggest that blocking this interaction may have therapeutic potential in treating HSV-1-associated eye disease.
IMPORTANCE Glycoprotein K (gK) is an essential and highly conserved HSV-1 protein. Previously we reported that gK binds to SPP, an endoplasmic reticulum (ER) protein, and blocking this binding reduces virus infectivity in vitro and also affects gK and UL20 subcellular localization. To evaluate the function of gK binding to SPP in vivo, we generated SPP inducible knockout mice and found that in the absence of SPP: (1) Significantly less HSV-1 replication was seen in ocularly infected mice than in control mice; (2) Expression of various HSV-1 genes and cellular infiltrates in the eye and trigeminal ganglia of infected mice was less than in control mice; and (3) Latency was significantly reduced in infected mice. Thus, blocking of gK binding to SPP may be a useful tool to control HSV-1-induced eye disease in patients with herpes stromal keratitis (HSK).
Functional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here we take an alternative approach to map functional constraints on Zika virus's envelope (E) protein by using deep mutational scanning to measure how all amino-acid mutations to the protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function, but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein, and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution.
IMPORTANCE Zika virus has recently been shown to be associated with severe birth defects. The virus's E protein mediates its ability to infect cells, and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino-acid mutations to E protein affected the virus's ability to grow in cells in the lab and escape from several antibodies. The resulting maps relate changes in the E protein's sequence to changes in viral function, and therefore provide a valuable complement to existing maps of the physical structure of the protein.
Type I and type III interferons (IFN) can promote adaptive immune responses in mice and improve vaccine-induced resistance to viral infections. The adjuvant effect of type III IFN (IFN-) specifically boosts mucosal immunity by an indirect mechanism, involving IFN--induced production of thymic stromal lymphopoietin (TSLP), a cytokine that activates immune cells. To date it remained unclear whether the previously described adjuvant effect of type I IFN (IFN-aalpha;/bbeta;) would also depend on TSLP and whether type I IFN stimulates different antibody subtypes. Here we show that after infection with a live attenuated influenza virus, mice lacking functional type I IFN receptors failed to produce normal amounts of virus-specific IgG2c and IgA antibodies. In contrast, mice lacking functional IFN- receptors contained normal levels of virus-specific IgG2c but had reduced IgG1 and IgA antibody levels. When applied together with protein antigen, IFN-aalpha; stimulated the production of antigen-specific IgA and IgG2c to a greater extent than IgG1, irrespective of whether the mice expressed functional TSLP receptors and irrespective of whether the vaccine was applied by the intranasal or the intraperitoneal route. Taken together, these results demonstrate that the adjuvant activities of type I and type III IFNs are mechanistically distinct.
IMPORTANCE Interferons can shape antiviral immune responses, but it is not understood well how they influence vaccine efficacy. We find that type I IFN preferentially promotes the production of antigen-specific IgG2c and IgA antibodies after infection with a live attenuated influenza virus or after immunization with influenza subunit vaccines. By contrast, type III IFN specifically enhances influenza virus-specific IgG1 and IgA production. The adjuvant effect of type I IFN was not dependent on TSLP which is essential for the adjuvant effect of type III IFN. Type I IFN boosted vaccine-induced antibody production after immunization by the intranasal or the intraperitoneal route, whereas type III IFN exhibited its adjuvant activity only when the vaccine was delivered by the mucosal route. Our findings demonstrate that type I and type III IFNs trigger distinct pathways to enhance the efficacy of vaccines. This knowledge might be used to design more efficient vaccines against infectious diseases.
Several studies support a role for specific Killer Immunoglobulin-like Receptor (KIR)/HLA combinations in protection from HIV infection and slower time to AIDS. NK cells acquire effector functions through education, a process that requires the interaction of inhibitory Natural Killer (NK) cell receptors with their major histocompatibility complex (MHC) class I (or HLA-I) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2 and KIR2DL3 whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of cell surface HLA-A, B and C on infected CD4 T cells (iCD4). Here, we investigated whether education through iKIR/HLA interactions influenced NK responses to autologous iCD4. Enriched NK cells were stimulated with autologous iCD4 or uninfected CD4 cells as controls. The capacity of NK cells to produce CCL4, IFN- and/or CD107a by single positive (sp) KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1 NK cells was assessed by flow cytometry. Overall, we observed that NK cell education potency was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of their cognate HLA-ligand on autologous iCD4 as measured by the frequency of spiKIR+ NK cells producing CCL4, IFN- and/or CD107a. Both NK cell education and HIV mediated changes in HLA expression influenced NK responses to iCD4 cells.
IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK and autologous HIV-infected cells to examine the role of education through four inhibitory Killer Immunoglobulin Receptors (iKIRs) from persons having HLA types able to educate, versus not, NK cells bearing one of these iKIRS. HIV-infected cells activated NK cells through missing-self mechanisms due HIV Nef and Vpu mediated downmodulation of cell surface HLA expression. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV+ cells by producing CCL4, IFN- and CD107a. As NK cells were from HIV uninfected individuals, they model the consequences of healthy NK/HIV+ cell interactions occurring in the HIV eclipse phase when new infections are susceptible to extinction.
Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation suggesting these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: Firehammervirus and Fletchervirus. Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are substituted with modified bases. Fletcherviruses substitute dG with 2'-deoxyinosine, while the Firehammerviruses substitute dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), non-canonical nucleotides previously described, but never observed making a 100% base substitution in a virus. We analyzed genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these non-canonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages, and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG-phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.
IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain non-canonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been under-reported. To date, two genomic guanosine modifications have been observed in phage genomes, both low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which substitute all guanosine bases in the genome in a genus-specific manner.
The RIG-I-like receptors (RLRs) are double-stranded RNA binding proteins that play a role in initiating and modulating cell intrinsic immunity through the recognition of RNA features typically absent from the host transcriptome. While initially characterized in the context of RNA virus infection, evidence has now accumulated establishing the role of RLRs in DNA virus infection. Here, we review recent advances in RLR-mediated restriction of DNA virus infection with an emphasis on the RLR ligands sensed.
Although vertical transmission from parents-to-offspring through seeds is an important fitness component of many plant viruses, very little is known about the factors affecting this process. Viruses reach the seed by direct invasion of the embryo and/or by infection of the ovules or the pollen. Thus, it can be expected that the efficiency of seed transmission would be determined by: (i) virus within-host multiplication and movement, (ii) virus ability to invade gametic tissues, (iii) plant seed production upon infection, and (iv) seed survival in the presence of the virus. However, these predictions have been seldom experimentally tested. To address this question, we have challenged 18 Arabidopsis thaliana accessions with Turnip mosaic virus and Cucumber mosaic virus. Using these plant-virus interactions, we analysed the relationship between the effect of virus infection on rosette and inflorescence weights, on short-, medium- and long-term seed survival, virulence, number of seeds produced per plant, virus within-host speed of movement, virus accumulation in the rosette and inflorescence, and the efficiency of seed transmission measured as per cent and as total number of infected seeds. Our results indicate that the best estimators of per cent seed transmission are the within-host speed of movement and multiplication in the inflorescence. Together with these two infection traits, virulence and the number of seeds produced per infected plant were also associated with the number of infected seeds. Our results provide support for theoretical predictions and contribute to understand the determinants of a process central to plant-virus interactions.
IMPORTANCE One of the major contributing factors to plant virus long-distance dispersal is the global trade of seeds. This is because more than 25% of plant viruses can infect seeds, which are the main mode of germplasm exchange/storage, and start new epidemics in areas where they were not previously present. Despite of the relevance of this process for virus epidemiology and disease emergence, the infection traits associated with the efficiency of virus seed transmission are largely unknown. Using turnip mosaic and cucumber mosaic viruses and their natural host Arabidopsis thaliana as model systems, we have identified the within-host speed of virus colonization and multiplication in the reproductive structures as main determinants of the efficiency of seed transmission. These results contribute to shed light on the mechanisms by which plant viruses disperse and optimize their fitness, and may help to design more efficient strategies to prevent seed infection.
Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus, but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variation of a human H1N1 pandemic virus, a seasonal H3N2 virus, and a H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with that seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.
SIGNIFICANCE Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals, but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza pathology, host passage evolution will vary depending on the specific virus tested.
The nonstructural protein NS5A of the hepatitis C virus (HCV) is a phosphorylated protein indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from hypo- to hyper-phosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyper-phosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyper-phosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyper-phosphorylated states and intracellular distribution necessary for the HCV life cycle.
IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyper-phosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting one as phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must stay wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyper-phosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyper-phosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyper-phosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.
Type I interferons (IFNs), including IFN-aalpha; and IFN-bbeta;, potently suppress HIV-1 replication by up-regulating IFN-stimulated genes (ISGs). The viral capsid protein (CA) partly determines the sensitivity of HIV-1 to IFNs. However, it remains to be determined whether CA-related functions including utilization of known host factors, reverse transcription, and uncoating affect sensitivity of HIV-1 to IFNs-mediated restriction. Recently, we identified an HIV-1 CA variant that is unusually sensitive to IFNs. This variant, called RGDA/Q112D, contains multiple mutations in CA: (H87R, A88G, P90D, P93A and Q112D). To investigate how an IFN-hypersensitive virus can evolve to overcome IFN-bbeta;-mediated blocks targeting the viral capsid, we adapted the RGDA/Q112D virus in IFN-bbeta;-treated cells. We successfully isolated IFN-bbeta; resistant viruses, which contain either a single Q4R substitution or the double amino acid change G94D/G116R. These two IFN-bbeta; resistance mutations variably changed in sensitivity to MxB, CPSF6, and CypA indicating that the observed loss of sensitivity was not due to interactions with these known host CA interacting factors. In contrast, the two mutations apparently functioned through distinct mechanisms. The Q4R mutation dramatically accelerated the kinetics of reverse transcription and initiation of uncoating of the RGDA/Q112D virus in the presence or absence of IFN-bbeta;, whereas the G94D/G116R mutations affected the reverse transcription only in the presence of IFN-bbeta;, most consistent with a mechanism of disrupting binding to an unknown IFN-bbeta; regulated host factor. These results suggest that HIV-1 can exploit multiple, known host factors-independent pathways to avoid IFN-bbeta;-mediated restriction by altering capsid sequences and subsequent biological properties.
Importance HIV-1 infection causes robust innate immune activation in virus-infected patients. This immune activation is characterized by elevated levels of type I interferons (IFNs), which can block HIV-1 replication. Recent studies suggest that the viral capsid protein (CA) is a determinant for sensitivity of HIV-1 to IFN-mediated restriction. Specifically, it was reported that loss of CA interactions with CPSF6 or CypA leads to higher IFN sensitivity. However, the molecular mechanism of CA adaptation to IFNs sensitivity is largely unknown. Here, we experimentally evolved an IFN-bbeta;-hypersensitive CA mutant, which shows decreased binding to CPSF6 and CypA, in IFN-bbeta;-treated cells. The CA mutations that emerged from this adaption indeed conferred IFN-bbeta; resistance. Our genetic assays suggest limited contribution of known host factors for IFN-bbeta; resistance. Strikingly, one of these mutations accelerated the kinetics of reverse transcription and uncoating. Our findings suggest that HIV-1 selected multiple, known host factor-independent pathways to avoid IFN-bbeta;-mediated restriction.
Circoviruses are the smallest DNA viruses known to infect mammals and avian species. Although circoviruses are known to be associated with a range of clinical diseases, details in circovirus DNA release still remain unknown. Herein, we identified p32 as a key regulator for porcine circoviral nuclear egress. Upon porcine circovirus type 2 (PCV2) infection, p32 was recruited into nucleus by the viral Cap protein; simultaneously, PKC- was phosphorylated at threonine 505 by PLC-mediated signaling at the early state of infection, which was further amplified by JNK and ERK signaling at late infection phase. p32 functioned as an adaptor to recruit phosphorylated PKC- and Cap to the nuclear membrane to phosphorylate lamin A/C, resulting in a rearrangement of nuclear lamina thus facilitated viral nuclear egress. Consistent with these findings, knockout of p32 in PCV2-infected cells markedly reduced the phosphorylation of PKC- and impeded the recruitment of p-PKC- and Cap to the nuclear membrane, hence abolishing the phosphorylation of lamin A/C and nuclear lamina rearrangement. As a result, p32 depletion profoundly impaired the production of cell-free viruses during PCV2 infection. We further identified the N-terminal llsquo;24RRR26' of Cap to be crucial for binding to p32, and mutation of these three arginine residues significantly weakened the replication and pathogenesis of PCV2 in vivo. In summary, our findings highlight a critical role of p32 in activation and recruitment of PKC- to phosphorylate lamin A/C and facilitate porcine circoviral nuclear egress, and certainly help understanding the mechanism of PCV2 replication.
IMPORTANCE Circovirus infections are highly prevalent in mammalian and avian species. Circoviral capsid protein is the only structural protein of the virion that plays an essential role in viral assembling. However, the machinery of circovirus nuclear egress is currently unknown. In this work, we identified p32 as a key regulator of PCV2 nuclear egress which forms complex with the viral Cap protein to enhance PKC- activity; this resulted in a recruitment of phosphorylated PKC- to the nuclear membrane, which further phosphorylates lamin A/C to promote the rearrangement of nuclear lamina and facilitate viral nuclear egress. Notably, we found that the N-terminal llsquo;24RRR26rrsquo; of Cap, a highly conserved motif among circovirus species, was required for interacting with p32, and that mutation of this motif markedly impeded PCV2 nuclear egress. These data indicate that p32 is a critical regulator of PCV2 nuclear egress and reveal the importance of this finding in circovirus replication.
Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome, on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa non-structural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on a SDS-PAGE gel and a handful of proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pull-down assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses.
IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward firstly understanding how the viruses survive the plant defense mechanisms, and secondly gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses including Bamboo mosaic virus, Foxtail mosaic virus and Potato virus X. Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.
Maternal vaccination may be the most effective and safest approach to protection of infants from respiratory syncytial virus (RSV) infection, a severe acute lower respiratory track disease in infants and young children worldwide. We have previously compared five different virus-like particle (VLP) associated, mutation stabilized pre-fusion F proteins, including the prototype DS-Cav1 F VLPs. We showed that alternative versions of pre-fusion F proteins have different conformations and induce different populations of anti-F protein antibodies. Two of these alternative pre-F VLPs, UC-2 F and UC-3 F VLPs, stimulated, in mice, higher titers of neutralizing antibodies than DS-Cav1 F VLPs (Vaccines 7:21). Here we describe comparison of these two pre-F VLPs with DS-Cav1 F VLPs as maternal vaccines in cotton rats and report that UC-3 F VLPs significantly increased neutralizing antibody titers in pregnant dams compared to DS-Cav1 F VLPs. The neutralizing antibody titers in sera of offspring of dams immunized with UC-3 F VLPs were significantly higher than in offspring of dams immunized with DS-Cav-1 VLPs. This increase in serum NAbs translated to a 6 to 40-fold lower virus titer in lungs of RSV challenged offspring of dams immunized with UC-3 F VLPs compared to offspring of dams immunized with DS-Cav1 F VLPs. Importantly, offspring of UC-3 F VLP immunized dams showed significant protection from lung pathology and from induction of inflammatory lung cytokine mRNA expression after RSV challenge. Immunization with UC-3 F VLPs also induced durable levels of high titer neutralizing antibodies in dams.
SIGNIFICANCE Respiratory syncytial virus (RSV) is a significant human pathogen severely impacting neonates and young children, but no vaccine exists to protect this vulnerable population. Furthermore, direct vaccination of neonates is likely ineffective due to the immaturity of their immune system, and neonate immunization is potentially unsafe. Maternal vaccination may be the best and safest approach to protection of neonates through the passive transfer of maternal neutralizing antibodies in utero to the fetus after maternal immunization. Here we report that immunization of pregnant cotton rats, a surrogate model for human maternal immunization, with novel RSV virus-like particle (VLP) vaccine candidates containing stabilized pre-fusion RSV F proteins provide significant levels of protection of offspring of immunized dams from RSV challenge. We also found that antibodies induced by VLPs containing different versions of the pre-fusion F protein varied by 40-fold in the extent of protection provided to offspring of vaccinated dams upon RSV challenge.
The HIV-1 Gag matrix (MA) domain mediates localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25/26 or 29/31: Lys-ggt;Arg (KR) (25/26KR and 29/31KR) and Lys-ggt;Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently unlike the corresponding KR mutants. Both 25/26KT Gag-YFP and 29/31KT Gag-YFP bound non-specifically to PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of non-specific membrane binding.
IMPORTANCE The PM-specific localization of HIV-1 Gag is a crucial early step in the infectious progeny production. The interaction between the MA highly basic region (HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents non-specific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2-binding ability is sufficient for MA to direct Gag specifically to the PM. The current study revealed for the first time an excellent correlation between RNA binding of MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provided cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures specific localization of Gag to the PM.
Human cytomegalovirus (HCMV) can cause congenital infection which is a leading cause of childhood disabilities. Since the rate of maternal-fetal transmission is much lower in naturally infected (HCMV-seropositive) women, we hypothesize that a vaccine candidate capable of eliciting immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV. We have previously described a replication-defective virus vaccine based on AD169 strain (Wang et al., Sci Transl Med 8:362ra145, 2016). The vaccine, named V160, has been shown to be safe and immunogenic in HCMV-seronegative human subjects, eliciting both humoral and cellular immune responses (Adler et al., JID, 220:411-9, 2019). Here we further showed that sera from V160-immunized HCMV-seronegative subjects shared similar quality attributes to those from seropositive subjects, including high avidity antibodies to viral antigens, coverage against a panel of genetically distinct clinical isolates, and protection against viral infection in diverse types of human cells in culture. More importantly, vaccination appeared efficient in priming the human immune system, inducing memory B-cells in six V160 recipients at frequencies comparable to those of three HCMV-seropositive subjects. Our results demonstrate the ability of V160 to induce robust and durable humoral memory responses to HCMV, justifying further clinical evaluation of the vaccine against congenital HCMV.
IMPORTANCE In utero HCMV infection can lead to miscarriage or childhood disabilities, and an effective vaccine is urgently needed. Since children born to women who are seropositive prior to pregnancy are less likely to be affected by congenital HCMV, it has been hypothesized that a vaccine capable of inducing an immune response resembling those in HCMV-seropositive women may be effective. We previously described a replication-defective virus vaccine that has been demonstrated safe and immunogenic in HCMV-seronegative subjects. Here we conducted additional analyses to show that the vaccine can induce antibodies with functional attributes similar to those from HCMV-seropositive subjects. Importantly, vaccination can induce long lived memory B-cells at frequencies comparable to those seen in HCMV-seropositive subjects. We conclude that this vaccine is a promising candidate that warrants further clinical evaluation for prevention of congenital HCMV.
A vaccine against congenital cytomegalovirus (cCMV) is a high priority. The guinea pig is a small animal model for cCMV. A
IMPORTANCE CMV is a leading cause of congenital disease in newborns and an effective vaccine remains an elusive goal. The guinea pig is the only small animal model for cCMV. Guinea pig cytomegalovirus (GPCMV) encodes a glycoprotein pentamer complex (PC) for cell entry into non-fibroblast cells, including placental trophoblasts to enable cCMV. As with HCMV, GPCMV uses a specific cell receptor (PDGFRA) for fibroblast entry but other receptors are required for non-fibroblast cells. A disabled infectious single-cycle (DISC) GPCMV vaccine strain induced an antibody immune response to the viral pentamer to enhance virus neutralization on non-fibroblast cells and vaccinated animals were fully protected against cCMV. Inclusion of the PC as part of a vaccine design dramatically improved vaccine efficacy and underlines the importance of the immune response to the PC in contributing towards protection against cCMV. This vaccine represents an important milestone in the development of a vaccine against cCMV.
CD4 downregulation on infected cells is a highly conserved function of primate lentiviruses. It has been shown to positively impact viral replication by a variety of mechanisms including enhanced viral release and infectivity, decrease of cell reinfection and protection from antibody-dependent cellular cytotoxicity (ADCC), which is often mediated by antibodies that require CD4 to change envelope (Env) conformation. Here we report that incorporation of CD4 into HIV-1 viral particles affects Env conformation resulting in the exposure of occluded epitopes recognized by CD4-induced antibodies. This translates into enhanced neutralization susceptibility by these otherwise non-neutralizing antibodies but is prevented by the HIV-1 Nef accessory protein. Altogether, these findings suggest that another functional consequence of Nef-mediated CD4 downregulation is the protection of viral particles from neutralization by commonly-elicited CD4-induced antibodies.
IMPORTANCE It has been well established that Env-CD4 complexes expose epitopes recognized by commonly-elicited CD4-induced antibodies at the surface of HIV-1-infected cells, rendering them vulnerable to ADCC responses. Here we show that CD4 incorporation has a profound impact on Env conformation at the surface of viral particles. Incorporated CD4 exposes CD4-induced epitopes on Env, rendering HIV-1 susceptible to neutralization by otherwise non-neutralizing antibodies.
Mink enteritis virus (MEV), an autonomous parvovirus, causes acute hemorrhagic enteritis in minks. The molecular pathogenesis of MEV infection has not been fully understood. In this study, we observed a significantly increased apoptosis in the esophagus, small intestine, mesenteric lymph nodes, and kidney in minks experimentally infected with MEVB strain (MEVB). In vitro infection of MEVB in feline F81 cells decreased cell viability, induced cell cycle arrest at G1 phase, and apoptosis. By screening MEV nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2), we demonstrated that the MEV NS1 induced apoptosis in both F81 and human embryonic kidney (HEK) 293T cells, similar to that induced during MEV infection in minks. We found that the NS1 protein induced apoptosis in HEK293T cells was not mediated by the death receptor but the mitochondrial pathway, as demonstrated by mitochondrial depolarization, opening of mitochondrial transition pore, release of cytochrome C, and activation of caspase-9 and -3. Moreover, in NS1-transfected cells, we observed an increase of Bax expression and its translocation to the mitochondria, as well as an increased ratio of the Bax/Bcl-2, ROS production, and activated p38 MAPK and p53. Taken together, our results demonstrated that MEV induces apoptosis through activation of p38 MAPK and the p53-mediated mitochondrial apoptotic pathway induced by NS1 protein, which sheds light on the molecular pathogenesis of MEV-infection.
MEV causes fatal hemorrhagic enteritis in minks. Apoptosis is a cellular mechanism that effectively sacrifices virus-infected cells to maintain homeostasis between the virus and host. In this study, we demonstrated that MEV induces apoptosis both in vivo and in vitro. Mechanistically, the viral large nonstructural protein NS1 activates p38 MAPK that leads p53 phosphorylation to mediate the mitochondrial apoptotic pathway, but not the death receptor-mediated apoptotic pathway. This is the first report to uncover the mechanism underlying MEV-induced apoptosis.
Respiratory syncytial virus (RSV) infects and causes disease in infants and re-infects with reduced disease throughout life without significant antigenic change. In contrast, re-infection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV re-infections. We therefore compared the ability of RSV and IAV to activate primary human cord-blood (CB) and adult blood (AB) myeloid (m)DC. While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase maturation markers CD38 and CD86, or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently up-regulating activation markers and cytokine production, IAV-induced CCR5 down-regulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection.
IMPORTANCE Respiratory syncytial virus (RSV) causes disease early in life and can re-infect symptomatically throughout life without undergoing significant antigenic change. In contrast, re-infection by influenza A virus (IAV) requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC). We used myeloid (m)DC from cord blood and adult blood donors to evaluate whether immunological immaturity contributes to the inability to mount a fully protective immune response to RSV. While IAV induced some activation and chemokine receptor switching in cord blood mDC, RSV did not. This appeared to be due to a lack of activation and a weak and mostly reversible inhibition of DC functions. Both viruses induced a stronger activation in mDC from adults. Thus, inefficient stimulation of mDC by RSV and immunological immaturity may contribute to reduced immune responses and increased susceptibility to RSV disease and re-infection in young infants.
H3N2 strains of influenza A virus emerged in humans in 1968 and have continued to circulate, evolving in response to human immune pressure. During this process of "antigenic drift," viruses have progressively lost the ability to agglutinate erythrocytes of various species and to replicate efficiently under the established conditions for amplifying clinical isolates and generating vaccine candidates. We have determined the glycome profiles of chicken and guinea pig erythrocytes to gain insights into reduced agglutination properties displayed by drifted strains and show that both contain complex sialylated N-glycans, but they differ with respect to the extent of branching, core fucosylation, and the abundance of poly-N-acetyllactosamine (PL) [-3Galbbeta;1-4GlcNAcbbeta;1-]n structures. We also examined binding of the H3N2 viruses using three different glycan microarrays: the synthetic Consortium for Functional Glycomics array, the defined N-glycan array designed to reveal contributions to binding based on sialic acid linkage type, branched structures, and core modifications, and the human lung shotgun glycan microarray. The results demonstrate that H3N2 viruses have progressively lost their capacity to bind nearly all canonical sialylated receptors other than a selection of bi-antennary structures and PL structures with or without sialic acid. Significantly, all viruses displayed robust binding to non-sialylated high mannose phosphorylated glycans, even as the recognition of sialylated structures is decreased through antigenic drift.
Influenza H3N2 subtype viruses have circulated in humans for over 50 years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of pre-existing immunity to previously circulating H3N2 strains. The changes in HA affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates.
Two viral non-structural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with HSP90 inhibitors, 17-AAG and ganetespib, suppressed RUBV genome replication. HSP90aalpha; physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrated that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication.
IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genome. However, host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrated that the HSP90 molecular chaperone is needed for efficient genome replication of RUBV. Further, we revealed that HSP90 interacts with RUBV non-structural protein p150, and its precursor polyprotein p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of non-structural proteins for RUBV genome replication. These findings provide novel insight into this hostnndash;viral interaction.
The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive understanding of how stable, uniform icosahedrons disassemble remains elusive, mainly due to the complexities involved in isolating transient intermediates. We utilized incremental heating to systematically characterize the disassembly pathway of a model non-enveloped virus, and identified an intriguing link between virus maturation and disassembly. Further, we isolated and characterized two intermediates by cryo-electron microscopy and 3D reconstruction, without imposing icosahedral symmetry. The first intermediate displayed a series of major, asymmetric alterations; while the second showed that the act of genome release, through the 2-fold axis, is actually confined to a small section on the capsid. Our study thus presents a comprehensive structural analysis of non-enveloped virus disassembly and emphasizes the asymmetric nature of programmed conformational changes.
IMPORTANCE Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by non-enveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates nndash; "eluted particles" and "puffed particles" of an insect nodavirus, Flock House Virus (FHV). Cryo-electron microscopy and 3D reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.
Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g. stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membrane-less metastable (shape-changing) condensates in the cytoplasm. In our studies we used the same cell lines and methods as used by previous investigators but concluded that wt MxA formed variably-sized spherical or irregular bodies, filaments and even a reticulum distinct from ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of GFP-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24pplusmn;0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase respectively in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-aalpha;-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature, and provide evidence for membrane-less MxA biomolecular condensates in the uninfected cell cytoplasm.
IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation, and further report that MxA forms membrane-less metastable (shape-changing) condensates in the cytoplasm consisting of variably-sized spherical or irregular bodies, filaments and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.
Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCVs strains can be propagated to high titers in standard non-human primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells which identified the coxsackie virus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that siRNA, anti-hCAR Mab RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9 targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.
IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays we identified and validated the coxsackie virus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs are both necessary to convert a non-susceptible cell line susceptible to ReCV infection. Follow up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.
The ER resident proteins, VAMP-associated protein (VAP) A and B (VAPA and VAPB) have been reported to be necessary for efficient HCV replication, but the specific mechanisms are not well understood. VAP proteins are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate (PI (4)P). Here we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI (4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP and the phosphatidylinositol 4-kinase, complete a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.
Hepatitis C Virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate (PI (4)P) in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol- binding protein (OSBP). OSBP exchanges PI (4)P with cholesterol, thus removing PI (4)P from the HCV RO. Here we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP may replenish PI at the HCV RO by interacting with VAMP associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, VAPs complete our understanding of the phosphoinositide cycle between ER and HCV RO.
Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyoma virus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein 1, which diminished virus binding to sialic acid-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated 1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231 and HS578T breast cancer cell lines recapitulated 1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers, and that breast cancer cells in by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of sialic acid (SA) levels on a panel of cancer cells showed that truncated 1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in 1 that prevents 1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, equivalent toxicity as wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data shows that tumor microenvironments have the potential to reduce infectivity of reovirus.
SIGNIFICANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have negative impact on proteinaceous cancer therapies such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.
Interferons (IFNs) play a crucial role in host antiviral response via activating JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen causing huge loss to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5 hours. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV non-structural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and coil-coil domain (CCD). Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.
SIGNIFICANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling as it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 as a mutation of it led to the loss of the capability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.
Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection ggt;10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.
IMPORTANCE In this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.
Several mammarenaviruses can cause deadly hemorrhagic fever infections in humans with limited preventative and therapeutic measures. Arenavirus cell entry is mediated by the viral glycoprotein (GP) complex that consists of the stable signal peptide SSP, the receptor-binding GP1 subunit, and the transmembrane GP2 subunit. The GP2 cytoplasmic tail (CT) is relatively conserved among arenaviruses and is known to interact with SSP to regulate GP processing and membrane fusion, but its biological role in the context of an infectious virus has not been fully characterized. Using a Pichinde virus (PICV) GP expression vector and a PICV reverse genetics system, we systematically characterized the functional roles of twelve conserved residues within GP2 CT in GP processing, trafficking, assembly, and fusion, as well as in viral replication. Except for P478A and K505A/R508A, alanine substitutions at all other conserved residues abolished GP processing and membrane fusion in plasmid-transfected cells. Six invariant H and C residues and W503 are essential for viral replication, as their mutant viruses could not be rescued. Both P480A and R482A mutant viruses were rescued, grew similarly as WT virus, and produced evidently processed GP1/GP2 subunits in virus-infected cells, despite the fact that the same mutations abolished GP processing and membrane fusion in a plasmid-based protein expression system, illustrating the importance of using an infectious virus system for analyzing viral glycoprotein function. In summary, our results demonstrate an essential biological role of GP2 CT in arenavirus replication and suggest it as a potential novel target for developing antivirals and/or attenuated viral vaccine candidates.
Several arenaviruses, such as Lassa virus (LASV), can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity with no FDA-approved vaccines or therapeutics. Viral entry is mediated by arenavirus GP that consists of the stable signal peptide (SSP), receptor-binding GP1, and transmembrane GP2 subunits. The cytoplasmic tail (CT) of GP2 is highly conserved among arenaviruses but its functional role in viral replication is not completely understood. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we show that the GP2 CT contains certain conserved residues that are essential for virus replication, which implicates it as a potentially good target for developing antivirals and live-attenuated viral vaccines against deadly arenavirus pathogens.
Hepatitis C virus (HCV) NS3 protein possesses protease and helicase activities and is considered an oncoprotein in the viral derived hepatocellular carcinoma. The NS3 associated oncogenesis has been studied but not fully understood. In this study, we have identified novel interactions of the NS3 protein with DNA repair factors, Werner syndrome protein (WRN) and Ku70, both in HCV subgenomic replicon system and in Huh7 cells expressing NS3. HCV NS3 protein inhibits WRN-mediated DNA repair and reduces the repair efficiency of non-homologous end joining. It interferes with Ku70 recruitment to the double strand break sites and alters the nuclear distribution of WRN-Ku repair complex. In addition, WRN is a substrate of the NS3-4A protease, the level of WRN protein is regulated by both proteasome degradation pathway and an HCV NS3-4A protease activity. The dual role of HCV NS3/NS3-4A proteins in regulating the function and expression level of the WRN protein intensifies the effect of impairment on DNA repair. This may lead to an accumulation of DNA mutations and genome instability, and eventually tumor development.
IMPORTANCE HCV infection is a worldwide problem of public health, and a major contributor to hepatocellular carcinoma. The single-stranded RNA virus undergoes high error rate of RNA-dependent RNA polymerase and develops strategies to escape immune system and hepatocarcinogenesis. Studies have revealed the involvement of HCV proteins in the impairment of DNA repair. The present study aimed to further elucidate mechanisms by which the viral NS3 protein impairs the repair of DNA damage. Our results clearly indicate that HCV NS3-4A protease targets WRN for degradation, meanwhile, diminishes the repair efficiency of non-homologous end joining by interfering with the recruitment of Ku protein to the DNA double strand break sites. The study describes a novel mechanism by which the NS3 protein influences DNA repair and provides new insight into the molecular mechanism of HCV pathogenesis.
Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by qPCR. Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses, and provide a robust platform for the development of novel antivirals.
IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.
Emerging studies demonstrate that the antiviral activity of viral fusion inhibitor peptides can be dramatically improved when being chemically or genetically anchored to the cell membrane, where viral entry occurs. We previously reported that short-peptide fusion inhibitor 2P23 and its lipid derivative possess highly potent antiviral activities against HIV-1, HIV-2, and simian immunodeficiency virus (SIV). To develop a sterilizing or functional cure strategy, here we genetically linked 2P23 and two control peptides (HIV-1 fusion inhibitor C34 and HBV entry inhibitor 4B10) with glycosylphosphatidylinositol (GPI) attachment signal. As expected, GPI-anchored inhibitors were efficiently expressed on the plasma membrane of transduced TZM-bl cells and primarily directed to the lipid raft site without interfering with the expression of CD4, CCR5 and CXCR4. GPI-anchored 2P23 (GPI-2P23) completely protected TZM-bl cells from infections of divergent HIV-1, HIV-2, and SIV isolates, as well as a panel of enfuvirtide (T20)-resistant mutants. GPI-2P23 also rendered the cells resistant to viral envelope-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission. Moreover, GPI-2P23 modified human CD4+ T cells (CEMss-CCR5) fully blocked both the R5- and X4-tropic HIV-1 isolates and displayed a robust survival advantage over the unmodified cells under HIV-1 infection. In contrast, it was found that GPI-anchored C34 was much less effective in inhibiting HIV-2, SIV, and T20-resistant HIV-1 mutants. Therefore, our studies have demonstrated that genetically anchoring a short-peptide fusion inhibitor to the target cell membrane is a viable strategy for gene therapy of both the HIV-1 and HIV-2 infections.
IMPORTANCE Antiretroviral therapy (ART) with multiple drugs in a combination can efficiently suppress HIV replication and dramatically reduces the morbidity and mortality associated with AIDS-related illness; however, ART cannot eradiate the HIV reservoirs, a lifelong treatment is required that often results in cumulative toxicities, drug resistance, and a multitude of complications, thus necessitating the development of sterilizing cure or functional cure strategies. Here we report that genetically anchoring the short-peptide fusion inhibitor 2P23 to the cell membrane can fully prevent infections from divergent HIV-1, HIV-2, and SIV isolates, as well as a panel of enfuvirtide-resistant mutants. The membrane-bound 2P23 also effectively blocks HIV-1 Env-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission, and renders CD4+ T cells non-permissive to the infection and a robust survival advantage over the unmodified cells. Thus, our studies verify a powerful strategy to generate resistant cells for gene therapy of both the HIV-1 and HIV-2 infections.
Upon replication in mucosal epithelia and transmission to nerve endings, capsids of herpes simplex virus type I (HSV-1) travel retrograde within axons to peripheral ganglia where life-long latent infections are established. A capsid-bound tegument protein, pUL37, is an essential effector of retrograde axonal transport and also houses a deamidase activity that antagonizes innate immune signaling. In this report, we examined whether the deamidase of HSV-1 pUL37 contributes to the neuroinvasive retrograde axonal transport mechanism. We conclude that neuroinvasion is enhanced by the deamidase, but the critical contribution of pUL37 to retrograde axonal transport functions independently of this activity.
Herpes simplex virus type 1 invades the nervous system by entering nerve endings and sustaining long-distance retrograde axonal transport to reach neuronal nuclei in ganglia of the peripheral nervous system. The incoming viral particle carries a deamidase activity on its surface that antagonizes antiviral responses. We examined the contribution of the deamidase to the hallmark neuroinvasive property of this virus.
Immune regulation of alphaherpesvirus latency and reactivation is critical for the control of virus pathogenesis. This is evident for herpes simplex virus type 1 (HSV-1), where cytotoxic T lymphocytes (CTLs) prevent viral reactivation, independent of apoptosis induction. This inhibition of HSV-1 reactivation has been attributed to granzyme B cleavage of HSV infected cell protein (ICP) 4, however it is unknown whether granzyme B cleavage of ICP4 can directly protect cells from CTL cytotoxicity. Varicella zoster virus (VZV) is closely related to HSV-1, however it is unknown whether VZV proteins contain granzyme B cleavage sites. Natural killer (NK) cells play a central role in VZV and HSV-1 pathogenesis and like CTLs utilize granzyme B to kill virally infected target cells. However whether alphaherpesvirus granzyme B cleavage sites could modulate NK cell mediated cytotoxicity has yet to be established. This study aimed to identify novel HSV-1 and VZV gene products with granzyme B cleavage sites and assess whether they could protect cells from NK cell mediated cytotoxicity. We have demonstrated that HSV ICP27, VZV ORF62 and VZV ORF4 are cleaved by granzyme B. However, in a NK cell cytotoxicity assay only VZV ORF4 conferred protection from NK cell mediated cytotoxicity. The granzyme B cleavage site in ORF4 was identified via site directed mutagenesis and surprisingly, the mutation of this cleavage site did not alter the ability of ORF4 to modulate NK cell cytotoxicity, suggesting that ORF4 has a novel immunoevasive function which is independent from the granzyme B cleavage site.
IMPORTANCE HSV-1 causes oral and genital herpes and establishes life-long latency in sensory ganglia. HSV-1 reactivates multiple times in a person's life and can cause life-threatening disease in immunocompromised patients. VZV is closely related to HSV-1 and causes chickenpox during primary infection and establishes life-long latency in ganglia from where it can reactivate to cause herpes zoster (shingles). Unlike HSV-1, VZV only infects humans and there are limited model systems, thus little is known concerning how VZV maintains latency and why VZV reactivates. Through studying the link between immune cell cytotoxic functions, granzyme B and viral gene products an increased understanding of viral pathogenesis will be achieved.
Attenuated poxviruses, like the modified vaccinia virus Ankara (MVA), are promising vectors for vaccines against infectious diseases and cancer. However, host innate immune responses interfere with the viral life cycle and also influence the immunogenicity of vaccine vectors. The sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a phosphohydrolase and reduces the cellular deoxynucleoside triphosphate (dNTP) concentration, which impairs poxviral DNA replication in human dendritic cells (DCs). The human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) encode an accessory protein called viral protein X (Vpx) that promotes proteasomal degradation of SAMHD1, leading to a rapid increase in cellular dNTP concentrations. To study the function of SAMHD1 during MVA infection of human DCs, the SIV vpx gene was introduced into the MVA genome (resulting in recombinant MVA-vpx). Infection of human DCs with MVA-vpx led to SAMHD1 protein degradation and enabled MVA-vpx to replicate its DNA genome and to express genes controlled by late promoters. Late gene expression by MVA-vpx might improve its vaccine vector property however unexpectedly type I interferon expression was blocked by vpx-expressing MVA. MVA-vpx can be used as a tool to study poxvirus host interaction and vector safety.
IMPORTANCE SAMHD1 is a phosphohydrolase and reduces cellular dNTP concentrations, which impairs poxviral DNA replication. The simian SIV accessory protein Vpx promotes degradation of SAMHD1, leading to increased cellular dNTP concentrations. Vpx addition enables poxviral DNA replication in human dendritic cells (DCs) and the expression of viral late proteins, which are normally blocked. SAMHD1 function during modified vaccinia virus Ankara (MVA) infection of human DCs was studied with recombinant MVA-vpx, expressing Vpx. Infection of human DCs with MVA-vpx decreased SAMHD1 protein amounts, enabling MVA DNA replication and expression of late viral genes. Unexpectedly, type I interferon expression was blocked after MVA-vpx infection. MVA-vpx might be a good tool to study SAMHD1 depletion during poxviral infections and provide insights into poxvirus host interaction.
Type III interferon (IFN), or IFN-, is an essential component of the innate immune response to mucosal viral infections. Both in the intestine and the lung, signaling via the IFN- receptor, IFNLR, controls clinically important viral pathogens including influenza virus, norovirus, and rotavirus. While it is thought that IFN- cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2-/-Ifnl3-/- mice, which lack all known functional murine IFN- cytokines. We demonstrate that, like Ifnlr1-/- mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus, and therefore genocopy Ifnlr1-/- mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-2 and IFN-3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens.
Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the type III interferon receptor, Ifnlr1, have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1. Our results support an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.
The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex, and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.
IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.
Hendra virus (HeV) is a zoonotic paramyxovirus that utilizes a trimeric fusion protein (F) within its lipid bilayer to mediate membrane merger with a cell membrane for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the pre-fusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a pre-fusion conformation-specific antibody prior to cell disruption, indicating that the introduced disulfide bonds did not significantly affect protein folding. This study is the first to report that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion.
The paramyxovirus Hendra virus (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion protein (F) may be applicable to a broad span of enveloped viruses. In this study, we demonstrate that introduced disulfide bonds between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still fold properly and bind a pre-fusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and expand our knowledge of the TMD and its role in HeV F stability and fusion promotion.
Equine Herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ORF2) was tested as a vaccine candidate. Three groups of horses (n=8 each) were infected intranasally with Ab4ORF2, the parent Ab4 virus, or kept as non-infected controls. Horses infected with Ab4ORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously non-infected horses (Control/Ab4) displayed clinical signs, shed high amounts of virus, and developed cell-associated viremia. In contrast, 5/8 and 3/8 horses previously infected with Ab4ORF2 or Ab4, respectively, were fully protected from challenge infection indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection and intranasal antibodies increased rapidly post challenge. Intranasal inflammatory markers were not detectable in protected horses, but quickly increased in Control/Ab4 horses during the first week after infection. Overall, our data suggest that pre-existing nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ORF2 as a vaccine candidate.
Importance Nasal EHV-1 shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination, emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to pre-existing local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies, and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.
Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: 1) an excessive amount of serum HBV virions containing immature genomes; 2) significantly reduced amounts of intracellular RC and SS DNAs; 3) less persistent intrahepatic and secreted HBV DNAs than the wild type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model.
IMPORTANCE. Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, no effective treatment can eradicate the virus from patients. HBV in chronic carriers does not exist as one single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. While the viral DNA of mutant I97L with immature genome is less persistent than wild type HBV in the time course experiments, surprisingly, viral DNA of mutant P130T with genome hypermaturation is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.
Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus in China and Southeast Asia with features of rapid spread, an expanding host range and cross-species transmission. The mechanisms of its infection and pathogenesis remain largely unclear. Here we investigated the tropism of this arbovirus in peripheral blood mononuclear cells of specific pathogen-free (SPF) ducks and SPF chickens and identified monocytes/macrophages as the key targets of TMUV infection. In vivo studies in SPF ducks and SPF chickens with monocyte/macrophage clearance demonstrated that the infection of monocytes/macrophages was crucial for viral replication, transmission and pathogenesis. Further genome-wide transcriptome analyses of TMUV-infected chicken macrophages revealed host antiviral innate immune barriers were the major targets of TMUV in macrophages. Despite the activation of major pattern-recognition receptor signalling, the inductions of IFN-aalpha; and IFN-bbeta; were blocked by TMUV infection on transcription and translation levels respectively. Meanwhile, TMUV inhibited host redox responses through repressing the transcription of genes encoding NADPH oxidase subunits and promoting Nrf2-mediated antioxidant responses. The recovery of either abovementioned innate immune barrier was sufficient to suppress TMUV infection. Collectively, we identify an essential step of TMUV infection and reveal extensive subversion of host antiviral innate immune responses.
Mosquito-borne flaviviruses include a group of pathogenic viruses that cause serious diseases in humans and animals, including dengue, West Nile, and Japanese encephalitis. These flaviviruses are zoonotic and use animals, including birds, as amplifying and reservoir hosts. Avian Tembusu virus (TMUV) is an emerging mosquito-borne flavivirus pathogenic for many avian species and can infect cells derived from mammals and humans in vitro. Although not currently pathogenic to primates, the infection of duck industry workers and the potential risk of TMUV infection in immunocompromised individuals have been highlighted. Thus, the prevention of TMUV in flocks is important for both avian and mammalian health. Our study reveals the escape of TMUV from the first line of the host defence system in the arthropod-borne transmission route of arboviruses, possibly helping to extend our understanding of flaviviruses infection in birds and refine the design of anti-TMUV therapeutics.
In mice, resistance to CNS disease induced by members of the genus Flavivirus is conferred by an allele of the 2'-5' oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses but amplification of viral proteins and double stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2'-5' linked oligoadenylates (2'-5'A) that interact with the latent endonuclease RNase L causing it to dimerize and cleave single stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L-/- mice. Evidence of RNase L activation in infected RNase L+/+ mice was indicated by higher levels of viral RNA in the brains of infected RNase L-/- mice. Activation of Type I IFN signaling was detected in both resistant and susceptible brains but Oas1a and Oas1b mRNA levels were lower in RNase L-/- mice of both types suggesting that activated RNase L may also have a pro-flaviviral effect. Inhibition of virus replication was robust in resistant RNase L-/- mice indicating that activated RNase L is not a critical factor in mediating this phenotype.
The mouse genome encodes a family of Oas proteins that synthesize 2'-5'A in response to dsRNA. 2'-5'A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L-/- mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo. Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L-/- mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L-/- mice indicating that activated RNase L may also have a pro-flaviviral affect. However, the resistance phenotype was equally robust in RNase L-/- and RNase L+/+ mice.
We present the genome sequences of Salmonella enterica tailed phages Sasha, Sergei, and Solent. These phages, along with Salmonella phages 9NA, FSL_SP-062 and FSL_SP-069 and the more distantly-related Proteus phage PmiS-Isfahan have similar sized genomes between 52 and 57 kbp in length that are largely syntenic. Their genomes also show substantial genome mosaicism relative to one another, which is common within tailed phage clusters. Their gene content ranges from 80 to 99 predicted genes, of which 40 are common to all seven and form the core genome which includes all identifiable virion assembly and DNA replication genes. The total number of gene types (pangenome) in the seven phages is 176, and 59 of these are unique to individual phages. Their core genomes are much more closely related to one another than to any other known phage, and they comprise a well-defined cluster within the family Siphoviridae. To begin to characterize this group of phages in more experimental detail, we identified the genes that encode the major virion proteins and examined the DNA packaging of the prototypic member, phage 9NA. We showed that it uses a pac site-directed headful packaging mechanism that results in virion chromosomes that are circularly permuted and about 13% terminally redundant. We also showed that its packaging series initiate with dsDNA cleavages that are scattered across a 170 bp region, and that its headful measuring device has a precision of pplusmn;1.8%.
IMPORTANCE The 9NA-like phages are clearly highly related to each other but are not closely related to any other known phage type. This work describes the genomes of three new 9NA-like phages, and experimental analysis of the proteome of the 9NA virion and DNA packaging into the 9NA phage head. There is increasing interest in the biology of phages because of their potential for use as antibacterial agents and for their ecological roles in bacterial communities. 9NA-like phages have been identified that infect two bacterial genera to date and related phages infecting additional Gram-negative hosts are likely to be found in the future. This work provides a foundation for the study of these phages which will facilitate their study and potential use.
In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single amino acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivo.
Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that a decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live-attenuated vaccine candidates.
The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 stain does not. Our data provides new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.
Importance A vaccine is still urgently needed to overcome the HCV epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis and eventually end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies, however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicates that HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.
HVTN 505 was a phase 2b efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) HIV vaccine regimen. Although the trial was stopped early for lack of overall efficacy, later correlates of risk and sieve analyses generated the hypothesis that the DNA/rAd5 vaccine regimen protected some vaccinees from HIV infection, yet enhanced HIV infection risk for others. Here we assessed whether and how host Fc gamma receptor (FcR) genetic variations influenced the DNA/rAd5 vaccine regimen's effect on HIV infection risk. We found that vaccine receipt significantly increased HIV acquisition compared with placebo receipt among participants carrying the FCGR2C-TATA haplotype (comprising minor alleles of four FCGR2C single nucleotide polymorphism (SNP) sites) (HR=9.79, p=0.035) but not among participants without the haplotype (HR=0.86, p=0.67); the interaction of vaccine and haplotype effect was significant (p=0.034). Similarly, vaccine receipt increased HIV acquisition compared with placebo receipt among participants carrying the FCGR3B-AGA haplotype (comprising minor alleles of the 3 FCGR3B SNPs) (HR=2.78, p=0.058) but not among participants without the haplotype (HR=0.73, p=0.44); again, the interaction of vaccine and haplotype was significant (p-value=0.047). The FCGR3B-AGA haplotype also influenced whether a combined Env-specific CD8+ T-cell polyfunctionality score and IgG response correlated significantly with HIV risk; an FCGR2A SNP and two FCGR2B SNPs influenced whether anti-gp140 antibody-dependent cellular phagocytosis correlated significantly with HIV risk. These results provide further evidence that Fc gamma receptor genetic variations may modulate HIV vaccine effects and immune function after HIV vaccination.
IMPORTANCE By analyzing data from the HVTN 505 efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) vaccine regimen, we found that host genetics, specifically Fc gamma receptor genetic variations, influenced whether receiving the DNA/rAd5 regimen was beneficial, neutral, or detrimental to an individual with respect to HIV-1 acquisition risk. Moreover, Fc gamma receptor genetic variations influenced immune responses to the DNA/rAd5 vaccine regimen. Thus, Fc gamma receptor genetic variations should be considered in the analysis of future HIV vaccine trials and the development of HIV vaccines.
Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative Caenorhabditis briggsae. Here we report on a collection of over 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Mělniiacute;k virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of co-infection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.
IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative C. briggsae and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Mělniiacute;k virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.
We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production, and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. The mechanism likely involves the interaction of IFI44L with the cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as the inhibitor of nuclear factor kappa B (IB) kinases (IKK) aalpha;, bbeta; and . Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3) and nuclear factor kappa-B (NF-B) inhibitor (IBaalpha;) mediated phosphorylation by IKK and IKKbbeta;, respectively. According to these results, IFI44L is a good target to treat diseases associated with excessive IFN and/or proinflammatory responses and for reducing viral replication.
IMPORTANCE Excessive innate immune responses could be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKK- aalpha;, bbeta; and . IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IBaalpha; mediated by IKK and IKKbbeta;, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.
Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok), and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (mAbs) and 38 Flucelvax-induced mAbs for avidity, cross reactivity and any selectivity towards the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of mAbs targeting epitopes near the receptor-binding domain on HA head (HAI+ mAbs) compared to Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive mAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive ASCs showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but may differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head.
IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax that use alternate production strategies, can induce protective influenza specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.
Lymph-borne Friend murine leukemia virus (FrMLV) exploits the sentinel macrophages in the draining popliteal lymph node (pLN) to infect highly permissive innate-like B-1 cells and establish infection in mice. The reason for FrMLV-sensitivity of B-1 cells and their impact on viral spread is unknown. Here we demonstrate that TLR7 sensing and type I interferon (IFN-I) signaling in B-1 cells contributes to FrMLV susceptibility. FrMLV infection in B-1 cell-deficient mice (bumble; IBNS-dysfunctional) was significantly lower than in the wild-type mice and was rescued by adoptive transfer of wild-type B-1 cells. This rescue of FrMLV infection in bumble mice was dependent on intact TLR7 sensing and IFN-I signaling within B-1 cells. Analyses of infected cell types revealed that the reduced infection in bumble mice was predominantly due to compromised virus spread to the B-2 cell population. Our data reveal how FrMLV exploits innate immune sensing and activation in the B-1 cell population for infection and subsequent spread to other lymphocytes.
IMPORTANCE Viruses establish infection in hosts by targeting highly permissive cell types. The retrovirus Friend murine leukemia virus (FrMLV) infects a subtype of B cells called B-1 cells that permit robust virus replication. The reason for their susceptibility had remained unknown. We found that innate sensing of incoming virus and the ensuing type I interferon response within B-1 cells is responsible for their observed susceptibility. Our data provide insights into how retroviruses coevolved with the host to coopt innate immune sensing pathways designed to fight virus infections for establishing infection. Understanding early events in viral spread can inform antiviral intervention strategies that prevent the colonization of a host.
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally-occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially-expressed in JSRV-infected lung compared to non-infected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and RT-qPCR. A key finding was the activation of anterior-gradient-2, yes-associated protein-1 and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity including genes encoding cytokines, chemokines and complement system proteins. In contrast, there was little evidence for upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.
IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.
Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression and parturition stressors did not lead to overt/recrudescent clinical disease but intermittent viremia and virus shedding were detected up to day 60 post-infection (pi) in all treatment groups following stress stimulation. Notably, real-time PCR, and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for dsRNA showed that SVA RNA persists in tonsillar cells in a double-stranded conformation. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 pi, confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.
IMPORTANCE Persistent viral infection infections have significant implications to disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally- or naturally infected animals long after resolution of the clinical disease. Here we showed that SVA establishes persistent infection in SVA infected animals with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as source of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology suggesting that persistently infected pigs may function as reservoirs for SVA.
Previously, we identified a set of lncRNAs that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the SeV- and IAV-induced activation of IFN-bbeta;. The luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-bbeta; reporter upon stimulation with RIG-I, MAVS, TBK1 or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. The CHIP assay showed that the enrichment of IRF3 and p65 at the IFN-bbeta; promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-bbeta; promoter. CHIRP, triplex pulldown and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-bbeta; promoter. Furthermore, the EMSA assay showed that lnc-MxA can form complexes with the IFN-bbeta; promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-bbeta; promoter to interfere with the activation of IFN-bbeta; transcription. Using a VSV infection assay, we confirmed that lnc-MxA can repress the RLR-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene that negatively regulates the transcription of IFN-bbeta; by forming an RNA-DNA triplex.
IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus known as a "cytokine storm" can also cause widespread tissue damage (Guo XZJ, Thomas PG. 2017. Seminars in Immunopathology 39:1-10; Yokota S. 2003. Nihon Rinsho 61:1953-1958; Clark IA. 2007. Immunology aamp; Cell Biology 85:271-273). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-bbeta; transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative feedback loop involved in maintaining immune homeostasis.
The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses against ASP in HIV-1 patients indicate that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows a polarized sub-nuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm, and is detectable on the cell surface even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 co-localize, suggesting that ASP might become incorporated in the membrane of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to anti-gp120 VRC01. Our studies indicate that ASP is an integral protein of the plasma membrane of chronically infected cells stimulated with PMA, and upon viral budding ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle, and suggest that ASP may represent a new therapeutic or vaccine target.
IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite or antisense direction of all other genes. The protein product of this antisense gene, called ASP, is poorly characterized and its role in viral replication remains still unknown. We provide evidence that the antisense protein, ASP of HIV-1 is found within the cell nucleus of unstimulated cells. In addition, we show that after PMA treatment ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrated that ASP is present on the surface of viral particles. Altogether, our studies identify ASP as a new structural component of the HIV-1 virus, and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surface of both infected cells and viral particles might be exploited therapeutically.
Due to the limiting coding capacity for members of the Picornaviridae family of positive-strand RNA viruses, their successful replication cycles require complex interactions with host cell functions. These interactions span from the down-modulation of many aspects of cellular metabolism to the hijacking of specific host functions used during viral translation, RNA replication, and other steps of infection by picornaviruses such as human rhinovirus, coxsackievirus, poliovirus, foot-and-mouth disease virus, enterovirus D-68, and a wide range of other human and non-human viruses. Although picornavirus replicate exclusively in the cytoplasm of infected cells, they have extensive interactions with host cell nuclei and the proteins and RNAs that normally reside in this compartment of the cell. This review will highlight some of the more recent studies that have revealed how picornavirus infections impact RNA metabolism of the host cell post-transcriptionally and how they usurp and modify host RNA binding proteins as well as microRNAs to potentiate viral replication.
Interferon (IFN) production activated by phosphorylated interferon regulatory factor 7 (IRF7) is a pivotal process during host antiviral infection. For viruses, suppressing the host IFN response is beneficial for viral proliferation; in such cases, evoking host-derived IFN negative regulators would be very useful for viruses. Here, we report that the zebrafish rapunzel 5 (RPZ5) protein which activated by virus degraded phosphorylated IRF7 is activated by TBK1, leading to the reduction of IFN production. Upon viral infection, zebrafish rpz5 was significantly upregulated as ifn, in response to the stimulation. Overexpression of RPZ5 blunted the IFN expression induced by both viral and retinoic acid-inducible gene I (RIG-I) likenndash;receptor (RLR) factors. Subsequently, RPZ5 interacted with RLRs but did not affect the stabilization of the proteins in the normal state. Interestingly, RPZ5 degraded the phosphorylated IRF7 under TBK1 activation through K48-linked ubiquitination. Finally, overexpression of RPZ5 remarkably reduced the host cell antiviral capacity. These findings suggest that zebrafish RPZ5 is a negative regulator of phosphorylated IRF7 and attenuates IFN expression during viral infection, providing insight into the IFN balance mechanism in fish.
IMPORTANCE The phosphorylation of IRF7 is helpful for host IFN production to defend against viral infection, thus, it is a potential target for viruses to mitigate the antiviral response. We report that the fish RPZ5 is an IFN negative regulator induced by fish viruses and degrades the phosphorylated IRF7 activated by TBK1, leading to IFN suppression and promoting viral proliferation. These findings reveal a novel mechanism for interactions between the host cell and viruses in the lower vertebrate.
In the host, many RING-domain E3 ligases have been reported to inhibit viral replication through various mechanisms. In a previous screen, we found that the porcine RING finger protein 114 (pRNF114), an RING-domain E3 ubiquitin ligase, inhibits classical swine fever virus (CSFV) replication. This study aimed to clarify the underlying antiviral mechanism of pRNF114 against CSFV. Upon CSFV infection, the pRNF114 mRNA was upregulated both in vitro and in vivo. CSFV replication was significantly suppressed in PK-pRNF114 cells stably expressing pRNF114 by lentivirus-delivered system, whereas CSFV growth was enhanced in PK-15 cells with RNF114 knockout by the CRISPR/Cas9 system. The RING domain of pRNF114, which has the E3 ubiquitin ligase activity, is crucial for its antiviral activity. Mechanistically, pRNF114 interacted with the CSFV NS4B protein through their C-terminal domains, which led to the K27-linked polyubiquitination and degradation of NS4B through a proteasome-dependent pathway. Collectively, these findings indicate that pRNF114 as a critical regulator of CSFV replication and uncover a mechanism by which pRNF114 employs its E3 ubiquitin ligase activity to inhibit CSFV replication.
IMPORTANCE The porcine RING finger protein 114 (pRNF114) is a member of RING-domain E3 ligases. In this study, pRNF114 is a potential anti-CSFV factor and the anti-CSFV effect of pRNF114 depends on its E3 ligase activity. Notably, pRNF114 targets and catalyzes the K27-linked polyubiquitination of the NS4B protein and then promotes proteasome-dependent degradation of NS4B, inhibiting the replication of CSFV. To our knowledge, pRNF114 is the first E3 ligase to be identified as being involved in anti-CSFV activity and targeting NS4B could be a crucial route for antiviral development.
The presence of T cell reservoirs in which HIV establishes latency by integrating into the host genome represents a major obstacle to a HIV cure and has prompted the development of strategies aimed at eradication of HIV from latently infected cells. The "Shock and kill" strategy is one of the most pursued approaches directed towards the elimination of viral reservoirs. Although several Latency-Reversing Agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. Here, we evaluated a novel immune-mediated approach to clear the HIV reservoir, based on the combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP and the FDA-approved histone deacetylase inhibitor Resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro. A reduction in HIV-harboring cells and in the total amount of HIV-DNA were also observed in CD4+ T central memory (TCM) cells, a primary cell model of latency, where Resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cellular-associated HIV-RNA were detected ex vivo in PBMCs and CD4+ T cells from individuals on suppressive ART. Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step towards HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation to reduce the viral reservoir and induce specific death of HIV-infected cells.
One of the challenges associated with HIV-1 infection is that, despite anti-retroviral therapies that reduce the levels of HIV-1 virus to undetectable levels, proviral DNA remains dormant in a sub-population of T lymphocytes. Numerous strategies to eliminate residual virus, so-called llsquo;shock and kill' strategies, have been proposed to reactivate latent virus and to eliminate the reservoir of HIV-1. In the present study, Palermo et al use a combination of small molecules that activate the innate anti-viral cGAS-STING immune response (di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV- infected T cell killing in cell lines, primary T lymphocytes and patient samples. These studies represent a novel strategy towards HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.
Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine, along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse genetics system (RGS) is urgently needed, but to date no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven plasmid-based system, and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV mini-genome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein complex (RNP), resulting in either attenuated or replication-incompetent viruses. Finally, by using the mini-genome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3' conserved noncoding region in IDV and ICV resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a mini-genome replication assay and a robust reverse genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.
IMPORTANCE Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs, and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse genetics system that can generate infectious viruses similar in replication kinetics to wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse genetics system and minireplicon reporter assay as described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.
Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: matrix (MA), capsid (CA), nucleocapsid (NC), and p6. Accurate and efficient cleavage of Gag is essential for virion infectivity: inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ~95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of the HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectibly affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired, and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially-processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions.
IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein resulted in strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that membrane binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.
Porcine reproductive and respiratory syndrome virus (PRRSV) is widely prevalent in pigs, resulting in significant economic losses worldwide. A compelling impact of PRRSV infection is severe pneumonia. In the present study, we found that IL-17 was up-regulated by PRRSV infection. Subsequently, we demonstrated that PI3K and p38MAPK signaling pathways were essential for PRRSV-induced IL-17 production as addition of PI3K and p38MAPK inhibitors dramatically reduced IL-17 production. Furthermore, we revealed that deleting C/EBPbbeta; and CREB binding motif in porcine IL-17 promoter abrogated its activation, and knock-down of C/EBPbbeta; and CREB remarkably impaired PRRSV-induced IL-17 production, suggesting that IL-17 expression was dependent on C/EBPbbeta; and CREB. More specifically, we demonstrated that PRRSV nonstructural protein 11 (nsp11) induced IL-17 production, which was also dependent on PI3K-p38MAPK-C/EBPbbeta;/CREB pathways. We then showed that Ser74 and Phe76 amino acids were essential for nsp11 to induce IL-17 production and viral rescue. In addition, IRAK1 was required for nsp11 to activate PI3K and enhance IL-17 expression by interacting with each other. Importantly, we demonstrated that PI3K inhibitor significantly suppressed IL-17 production and lung inflammation caused by HP-PRRSV in vivo, implicating that higher IL-17 level induced by HP-PRRSV might be associated with severe lung inflammation. These findings provide new insights onto the molecular mechanisms of the PRRSV-induced IL-17 production and help us further understand the pathogenesis of PRRSV infection.
IMPORTANCE Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) associated with severe pneumonia has been one of the most important viral pathogens in pigs. IL-17 is a pro-inflammatory cytokine that might be associated with the strong inflammation caused by PRRSV. Therefore, we seek to determine whether PRRSV infection affects IL-17 expression, and if so, it might partially explain the underlying mechanisms for the strong inflammation in HP-PRRSV-infected pigs, especially in lungs. Here we showed that PRRSV significantly induced IL-17 expression and subsequently we dissected the molecular mechanisms about how PRRSV regulated IL-17 production. Furthermore, we showed that Ser74 and Phe76 in nsp11 were indispensable for IL-17 production and viral replication. Importantly, we demonstrated that PI3K inhibitor impaired IL-17 production and alleviated lung inflammation caused by HP-PRRSV infection. Our findings will help us for a better understanding of PRRSV pathogenesis.
Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, that typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNPA1, hnRNPA2 and hnRNPB1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNPH1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro. HnRNPH1 depletion and mutation of a prominent viral RNA hnRNPH1 binding site decreased use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNPH1 through binding to its target element.
IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential, yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis acting elements regulating HIV-1 splicing.
An earlier report showed that HSV-1 expresses two miRNAs, miR-H28 and miR-H29, late in the infectious cycle. The mRNAs are packed in exosomes and in recipient cells restrict transmission of virus from infected to uninfected cells. We now report that (i) miR-H28 induces the synthesis of IFN both in infected cells and cells transfected with miR-H28, (ii) In infected cells IFN accumulates concurrently with viral proteins, (iii) IFN was produced in HEp-2 cells derived from cancer tissue and 293T cells derived from normal tissues, (iv) HSV-1 replication is affected by the exposure to IFN before infection but not during or after infection. The results presented in this report support the growing body of evidence that HSV-1 encodes function designed to reduce the spread of infection from infected to uninfected cells possibly in order to maximize transmission of virus from infected to uninfected individuals.
In this report we show that IFN is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN to curtail its spread from infected to uninfected cells. In essence this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.
Dengue virus (DENV) infection causes serious clinical symptoms, including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Vascular permeability change is the main feature of the diseases, and the abnormal expression of pro-inflammation cytokines is the important cause of vascular permeability change. However, the mechanism underlying vascular permeability induced by DENV is not fully elucidated. Here we reveal a distinct mechanism by which DENV infection promotes the NLRP3 inflammasome activation and interleukin-1 beta (IL-1bbeta;) release to induce endothelial permeability and vascular leakage in mice. DENV M protein interacts with NLRP3 to facilitate the NLRP3 inflammasome assemble and activation, which lead to the induction of pro-inflammation cytokine IL-1bbeta; activation and release. Notably, M can induce vascular leakage in mice tissues through activating NLRP3 inflammasome and IL-1bbeta;. More importantly, inflammatory cell infiltration and tissue injuries are induced by M in WT mice tissues, but they are not affected by M in NLRP3 knock-out (NLRP3-/-) mice tissues, and Evans blue intensities in WT mice tissues are significantly higher as compared with NLRP3-/- mice tissues, demonstrating an essential role of NLRP3 in M-induced vascular leakages in mice. Therefore, we propose that upon DENV infection, M interacts with NLRP3 to facilitate the inflammasome activation and IL-1bbeta; secretion, which lead to induction of endothelial permeability and vascular leakage in mice tissues. The important role of the DENV-M-NLRP3-IL-1bbeta; axis in the induction of vascular leakage provides new insights into the mechanisms underlying DENV pathogenesis and DENV-associated DHF and DSS development.
IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen and its infection is prevalent in over 100 tropical and sub-tropical countries or regions with approximately 2.5 billion people at risk. DENV infection induces a spectrum of clinical symptoms ranging from classical dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Therefore, it is important to understand the mechanisms underlying DENV pathogenesis. In this study, the authors reveal that the DENV membrane protein (M) interacts with the host NLRP3 protein to promote the NLRP3 inflammasome activation, which leads to the activation and release of pro-inflammation cytokine, interleukin-1 beta (IL-1bbeta;). More importantly, the authors demonstrate that M protein can induce vascular permeability and vascular leakage and NLRP3 is required for M-induced vascular leakage in mice tissues. Collectively, this study reveals a distinct mechanism underlying DENV pathogeneses and provides new insights into the development of therapeutic agent for DENV-associated diseases.
Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatio-temporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian States. The final dataset, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 States. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi. Within lineages II and III, seven and five sub-lineages, respectively, were distinguishable. The phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal towards the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast towards Taraba and Adamawa and south towards Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sub-lineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at higher resolution. In addition, we estimated the direction and timeframe of virus dispersal in the country.
IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sub-lineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows predicting into which geographical areas the virus might spread in future and prioritizing areas for Lassa fever surveillance. Our study not only aimed at generating Lassa virus sequences from across Nigeria, but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.
The innate immune response is vital for host defense and must be tightly controlled, but the mechanisms responsible for its negative regulation have not been fully understood. The cell growth-regulating nucleolar protein (LYAR) has been found to promote replication of multiple viruses in our previous study. Here, we report that LYAR acts as a negative regulator of the innate immune responses. We find that LYAR expression is induced by interferon-bbeta; (IFN-bbeta;) during virus infection. Further studies show that LYAR interacts with the phosphorylated IFN regulatory factor 3 (IRF3) to impede the DNA binding capacity of IRF3, thereby suppressing the transcription of IFN-bbeta; and the downstream IFN-stimulated genes (ISGs). In addition, LYAR inhibits nuclear factor-B (NF-B) mediated expression of proinflammatory cytokines. In summary, our study reveals the mechanism of LYAR modulating IFN-bbeta; mediated innate immune responses by targeting phosphorylated IRF3, which not only helps us to better understand the mechanisms of LYAR-regulated virus replication but also uncovers a novel role of LYAR in the host innate immunity.
IMPORTANCE IFN-I plays a critical role in the antiviral innate immune responses that protect host against virus infection. The negative regulators of IFN-I are not only important for fine-tuning the antiviral responses to pathogens but also for preventing excessive inflammation. Identification of negative regulators and study of their modulation in innate immune responses will lead to new strategies for the control of both viral and inflammatory diseases. Here, we report for the first time that LYAR behaves as a repressor of the host innate immune responses. We demonstrate that LYAR negatively regulates IFN-bbeta; mediated immune responses by inhibiting the DNA binding ability of IRF3. Our study reveals a common mechanism of LYAR promoting different virus replication and improves the knowledge of the host negative regulation of innate immune responses.
The plant-pathogenic virus, tomato spotted wilt virus (TSWV), encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1s revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication and plant-inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN. These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.
Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus, tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.
Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ~70-150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here we report the first complete knockout (KO) of viral ORF57 gene from all ~100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination of multiple rounds of cell selection and single cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ. We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells which contain only a few copies of KSHV genome. The ORF57-KO genome was stable in iSLK/Bac16 cells and upon lytic induction, could be partially rescued to express viral lytic gene ORF59 and production of infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high-copy numbers.
IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology could be applied to knock out the ORF57 gene from all ~100 copies of KSHV genome in PEL cells by co-expression of two gRNAs and Cas9 from a single expression vector in combination of single cell cloning. The gene knockout efficiency in this system could be evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology could also mediate ORF57 inversion in situ in the targeted site of KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57-KO genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of virus genome and to understand their contributions to viral genetics and virus life cycle.
Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication defective adenovirus (rAd) vaccine vectors are at the forefront of this work and tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV gag or other non-envelope proteins induce T cell responses that control virus load but disappointingly are unsuccessful so far in preventing infection and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomologus macaques, Macaca fascicularis (MCM), vaccinated against unmodified gag alone with a DNA prime followed by a rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection a significant reduction in peak virus load was observed in all vaccinated animals including those vaccinated with modified gag. These findings suggest that a non-persistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV-1 infection. The mechanisms are probably distinct from antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus infected cells and may be mediated in part by CD4 T cells.
IMPORTANCE The Simian Immunodeficiency Virus (SIV) macaque model represents the best animal model for testing new HIV-1 vaccines. Previous studies employing replication defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to envelope and neutralise the virus or mediate antibody dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established have also shown some success. By contrast the vaccine described here mediates resistance by a different mechanism from that described above which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.
During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) UL34 and UL31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro. Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein UL25, but not to the other capsid proteins tested (i.e., VP5, VP23 and UL17) and that HSV-1 NEC binding nucleocapsids was mediated by NEC interaction with UL25. UL31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and UL25. We also showed that alanine substitution of UL31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles, that were similar in size and morphology to primary envelopes, in the perinuclear space. These results suggested that NEC binding via UL31 R281 and D282 to nucleocapsids, probably to UL25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.
Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis has not been reported thus far. In this study, we have presented data showing that two amino acids in the membrane distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.
Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive, CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding, but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative, CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.
Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.
Human cytomegalovirus (HCMV) enters primary CD34+ hematopoietic progenitor cells by macropinocytosis where it establishes latency in part because its tegument transactivating protein, pp71, remains associated with endosomes and is therefore unable to initiate productive, lytic replication. Here we show that multiple HCMV strains also enter cell line models used to study latency by macropinocytosis and endocytosis. In all latency models tested, tegument-delivered pp71 was found co-localized with endosomal markers and not associated with the seven other cytoplasmic localization markers tested. Like the capsid-associated pp150 tegument protein, we detected capsid proteins initially associated with endosomes but later in the nucleus. Inhibitors of macropinocytosis and endocytosis reduced latent viral gene expression and precluded reactivation. Importantly, we utilized electron microscopy to observe entry by macropinocytosis and endocytosis, providing additional visual corroboration to our functional studies. Our demonstration that HCMV enters cell line models for latency in a manner indistinguishable from its entry into primary cells illustrates the utility of these cell lines for probing the mechanisms, host genetics, and small molecule-mediated inhibition of HCMV entry into the cell types where it establishes latency.
Primary cells cultured in vitro currently provide the highest available relevance for examining molecular and genetic requirements for the establishment, maintenance, and reactivation of HCMV latency. However, their expense, heterogeneity, and intransigence to both long-term culture and molecular or genetic modification create rigor and reproducibility challenges for HCMV latency studies. There are several cell line models for latency not obstructed by deficiencies inherent in primary cells. However, many researchers view cell line studies of latency as physiologically irrelevant because of the perception that these models display numerous and significant differences from primary cells. Here we show that the very first step in a latent HCMV infection, entry of the virus into cells, occurs in cell line models indistinguishably from how it occurs in primary CD34+ hematopoietic progenitor cells. Our data argue that experimental HCMV latency in cell lines and primary cells is much more similar than it is different.
Adeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies including a recent unbiased genomic screen have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surface of Crb3 KO cells, but not the universal AAV receptor (AAVR) leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.
Adeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome scale CRISPR knock out screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This work underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.
Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential to infection. While multiple host factors, including kinases and transcription factors, important to metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important to the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had similar levels of metabolites as those infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infectionmmdash;fatty acid elongase 7 (ELOVL7) and ER-stress related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells compared to WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV-infection, it is important to the remodeling of a subset of metabolic changes that occur during infection.
Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very long-chain fatty acids (VLCFAs). Further, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFAs accumulation that occurs in HCMV-infected cells. Our findings identify a viral protein that is important for changes to lipid metabolism that occurs following HCMV infection.
Influenza is a global public health problem. Current seasonal influenza vaccines have highly variable efficacy and hence attempts to develop broadly protective universal influenza vaccines with durable protection are underway. While much attention is given to the virus-related factors contributing to inconsistent vaccine responses, host-associated factors are often neglected. Growing evidences suggest that host factors including age, biological sex, pregnancy, and immune history play important roles as modifier of influenza virus vaccine efficacy. We hypothesize that host genetics, the hormonal milieu, and gut microbiota contribute to host-related differences in influenza virus vaccine efficacy. This review highlights the current insights and future perspectives into host-specific factors that impact influenza vaccine-induced immunity and protection. Consideration of the host factors that affect influenza vaccine-induced immunity might improve influenza vaccines by providing empirical evidence for optimizing or even personalizing vaccine type, dose, and use of adjuvants for current seasonal and future universal influenza vaccines.
Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets to host immune cells and causes life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a high virulent strain in vitro. The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate the infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs), and found that divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence lead to disparate mitochondrial function, morphology and metabolism. This in turn promoted distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34 infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in M1-polarized pro-inflammatory type transformation of macrophages and subsequently producing a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, decrease of mitochondrial membrane potential and impairment of electronic respiratory chain led to increased levels of apoptosis and ROS. These results are correlated with the viral pathogenicity loss and may help to understand the key mechanism of lentiviral attenuation.
Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how mitochondrial response changes in the cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in world. EIAVDLV34 is a parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after altering mitochondrial protein expression profile, EIAVDLV34 infected cells are transformed into M1-polarized type macrophages to cause inflammatory injury and the intrinsic apoptosis pathway is activated in EIAVDLV121 infected cells. These studies shed light on how the mitochondrial protein expression profile change from cells infected by pathogenic or attenuated lentivirus strains to drive different cellular response, especially from inflammation to apoptosis.
Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A and B virus (IAV/IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by as-yet undetermined protease(s).
Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII) and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9 and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited activation and spread of IBV in AECII, but did not affect IBV activation in HBEC and Calu-3 cells.
This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.
Influenza A and B viruses (IAV/IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site and a number of proteases have been shown to cleave HA in vitro. This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoire. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.
Herpes Simplex Virus type 1 (HSV-1) infects mucosal epithelial cells and establishes life-long infections in sensory neurons. Following reactivation, the virus is transferred anterograde to the initial site of infection or to sites innervated by infected neurons, causing vesicular lesions. Upon immunosuppression frequent HSV-1 reactivation can cause severe diseases such as blindness and encephalitis. Autophagy is a process whereby cell components are recycled, but it also serves as a defense mechanism against pathogens. HSV-1 is known to combat autophagy through the functions of the 134.5 protein, which prevents formation of the autophagophore by binding to Beclin-1, a key factor involved in the elongation of the isolation membrane, and by redirecting the protein phosphatase 1 aalpha; (PP1aalpha;) to dephosphorylate the translation initiation factor 2aalpha; (eIF2aalpha;) to prevent host translational shutoff. Other viral proteins that counteract innate immunity negatively impact autophagy. Here, we present a novel strategy of HSV-1 to evade the host, through the down-regulation of the autophagy adaptor protein sequestosome (p62/SQSTM1) and of the mitophagy adaptor optineurin (OPTN). This down-modulation occurs during the early steps of the infection. We also found that the Infected Cell Protein 0 (ICP0) of the virus mediates the down-modulation of the two autophagy adaptors in a mechanism independent of its E3 ubiquitin ligase activity. Cells depleted of either p62 or OPTN could mount greater antiviral responses, whereas cells expressing exogenous p62 displayed decreased virus yields. We conclude that down-regulation of p62/SQSTM1 and OPTN is a viral strategy to counteract the host.
Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves down-regulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question: Why does the virus target two major autophagy adaptors if it has mechanisms to block autophagosome formation? P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 could have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus could target another antiviral function of these proteins.
Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific pathogen free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed similar growth kinetics to the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h post-infection. Furthermore, we found that A72 cells derived from canine fibroblasts permit FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.
Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is lethal disease in cats. Tissue culture-adopted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that the type I FCoV strain C3663 efficiently induces FIP in specific pathogen free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.
Immune complex (IC) vaccines have been successfully used to increase immune responses against various pathogens, including HIV-1. Additionally, IC vaccines can induce qualitatively different antibody responses with distinct antigenic specificities compared to the same antigens used alone. Here we measured the HIV-1-specific antibody response in female New Zealand White rabbits after immunization with ICs made from BG505 SOSIP.664 trimers (BG505 trimers) and three different rabbit monoclonal antibodies (mAbs) with varying neutralization profiles. Two of the mAbs were specific for a hole in the glycan shield of the BG505 trimer while the third, which bound less avidly, was specific for determinants at the gp41/gp120 interface. We found that immunizing with one of the glycan hole-specific ICs resulted in lower levels of trimer-binding antibodies compared to vaccination with the uncomplexed trimer and that ICs made using either of the glycan hole-specific mAbs resulted in lower rates of anti-trimer antibody decay. We conclude that ICs based on mAbs that bound to the immunodominant glycan hole epitope likely diverted antibody responses, to some extent, away from this site and to other regions of the trimer. However, this outcome was not accompanied by a widening of the breadth or an increase in the potency of neutralizing antibody responses compared with uncomplexed trimers.
IMPORTANCE Immunodominant epitopes may suppress immune responses to more desirable determinants, such as those that elicit potentially protective neutralizing antibody responses. To overcome this problem, we attempted to mask immunodominant glycan holes by immunizing rabbits with immune complexes (ICs) consisting of the BG505 SOSIP.664 gp140 trimer and monoclonal antibodies that target the glycan holes. We found that IC vaccination likely diverted antibody responses, to some extent, away from glycan holes and toward other regions of the trimer. IC vaccination resulted in a slower decay of HIV-1-specific antibodies than did immunization with uncomplexed trimer. We did not observe a widening of the breadth or an increase in the potency of neutralizing antibody responses compared to uncomplexed trimers. Our results suggest that selective epitope dampening of BG505 trimers by ICs is rather ineffective. However, IC vaccination may represent a novel means of increasing the duration of vaccine-induced antibody responses.
Adenovirus (AdV)-based vectors are popular experimental vaccine vectors, but despite their ability to induce strong immune responses, their application is impeded by wide-spread pre-existing immunity against many AdV types that can impair or even abrogate induction of transgene-specific immune responses. Therefore, the development of vectors based on AdV types with low seroprevalence is important for effective AdV-based immunization in humans.
We investigated the immunization efficacy of vectors based on AdV types 48 and 50 in the ovalbumin (ova) model as well as the Friend retrovirus (FV) model, which allows testing the protective effect of vaccine-induced immunity. Using ova-encoding vectors, we found a significantly lower induction of ova-specific CD8+ T cells and antibody responses by Ad48- and Ad50-based vectors compared to Ad5. Similarly, we found a reduced induction of FV-specific CD8+ T cell responses in Ad48- and Ad50.Leader-Gag-immunized mice compared to Ad5; however, some of those mice were able to control the FV infection, and protection correlated with the level of neutralizing antibodies 10 days after FV challenge. Analyses of AdV-specific antibodies and CD8+ T cells induced by the individual AdV types revealed a high level of cross-reactivity, and the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice.
Our results show that immunity induced by Ad48- and Ad50-based vectors is reduced compared to Ad5, and is sufficient only in some of the immunized mice to control FV infection. A high level of cross-reactivity suggests that AdV pre-immunity must be considered even when applying rare AdV based vectors.
AdV-based vectors are important tools for the development of vaccines against a wide range of pathogens. While AdV vectors are generally considered safe and highly effective, their application can be severely impaired by pre-existing immunity due to wide-spread seroprevalence of some AdV types. The characterization of different AdV types with regard to immunogenicity and efficacy in challenge models is of great importance for the development of improved AdV-based vectors that allow for efficient immunization despite anti-AdV immunity. We show that immunity induced by an Ad48-based vector is inferior to Ad5, but can still mediate control of an FV infection in highly FV-susceptible mice. However, the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice. Importantly, we found cross-reactivity of both humoral and cellular immune responses raised by the individual AdV types, suggesting that switching to a different AdV type may not be sufficient to circumvent pre-existing anti-AdV immunity.
A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. Whilst much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as Modified Vaccinia Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because it contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.
Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here we find that the primacy of stable antigen is not generalisable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA, and so possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.
Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from a fast cytolysis causing echovirus 1 (EV1) with non-structural proteins from Coxsackievirus B5 (CVB5) showing persistent infection in RD cells. The results showed that the chimera behaved similar to the parental EV1 leading to efficient cytolysis in both permissive A549 and semi-permissive RD cells. In contrast to EV1 and chimera, CVB5 replicated slower in permissive cells and showed persistent infection in semi-permissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 virus batches constantly contained significant amounts of empty capsids, also in comparison to its close relative CVB3. During successive passaging of batch containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increased amounts of empty particles and lowering amounts of infective particles is dictated by the CVB5 structural proteins leading to slowing down the infection between passages. Furthermore, the key factor for persistent infection is the low amount of infective particles produced, not the high number of empty particles accumulating.
IMPORTANCE Enteroviruses cause several severe diseases with lytic infections that lead to rapid cell death but also persistent infections that are more silent, and lead to chronic states. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera between the structural proteins of echovirus 1 and non-structural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to production of high number of empty viruses (empty capsids), that do not contain genetic material and are unable to continue the infection. Coinciding with high number of empty capsids, also the amount of infective virions decreased. This characteristic property was not observed in the constructed chimeravirus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infection are essential in understanding the virus caused pathologies.
The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incoporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2), and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough mapping studies indicate that C-terminal CT helices are involved in MA binding, and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers.
The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood, but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interference with MA trimerization, and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.
Early HIV-1 treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in chronically SHIV-infected macaques, intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, here we used LSEVh-LS-F, a bispecific bnAb targeting CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically-infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.
IMPORTANCE Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since the early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for the treatment failure in the acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques, respectively. This study revealed the correlation between the fast clearance of infused bnAbs and the treatment failure during the acute period of infection.
Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of viral spike (S) protein to bind the host receptor, mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a), and mediate virus entry. Our previous crystal structure study of MHV NTD/mCEACAM1a complex (1) reveals that there are 14 residues in NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis, and identified I22 essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of S protein triggered either by receptor binding or high pH. Substitution of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from S protein, resulting in enhancement of high pH triggered receptor-independent syncytia (RIS) formation in 293T cells, compared to WT. By contrast, replacement of G29 with P, a turn prone residue with strict conformation, hindered virus entry and conformational changes of S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of NTD by G29P almost had no effect on pH-independent RIS induced by Y320A mutation in C-terminal domain (CTD) of S1 subunit, indicating that there might be absence of crosstalk between NTD and CTD during conformational changes of S protein. Our study will aid better understanding the mechanism how conformational changes of S protein is triggered.
IMPORTANCE Binding of MHV S protein to the receptor, mCEACAM1a, triggers the conformational changes of S proteins, leading to formation of six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of S protein is initiated after receptor binding has not been determined. In this study, we showed that, while replacement of G29, a residue at the edge of receptor binding interface and the center of structural turn after bbeta;1-sheet of S protein, with D or T triggered spontaneous conformational change of S protein and pH-independent RIS, G29P mutation significantly impeded the conformational changes of S proteins triggered either by receptor binding and pH 8.0. We reason that this structural turn might be critical for conformational change of S protein and altering this structural turn could initiate the conformational changes of S protein, leading to membrane fusion.
With a yearly death toll of 880,000, Hepatitis B Virus (HBV) remains a major health problem worldwide despite an effective vaccine and well-tolerated effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long term antiviral therapy and its integration nndash; though no longer able to support viral replication nndash; destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products and thus, is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity, but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.
We have previously demonstrated that Epstein-Barr virus DNA increases the production of the pro-inflammatory cytokine IL-17A in mice. This property may contribute to the established association between EBV and autoimmune diseases. The objective of the current study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To determine whether endosomal Toll-Like Receptors (TLRs) played a role in this pathway, the expression of TLR3, 7 or 9 was assessed by real-time RT-PCR in mouse spleens after injection of EBV DNA. Moreover, specific inhibitors were used for these TLRs in mouse Peripheral Blood Mononuclear Cells (PBMCs) cultured with EBV DNA and in mice injected with this viral DNA; IL-17A levels were then assessed using Enzyme Linked Immunosorbent Assay. The expression of the endosomal receptors TLR3, 7, and 9 was increased in mice injected with EBV DNA. When mouse immune cells were cultured with EBV DNA and a TLR3, 7 or 9 inhibitor or when mice were injected with the viral DNA along with either of these inhibitors, a significant decrease in IL-17A levels was detected. Therefore, endosomal TLRs are involved in the EBV DNA-mediated triggering of IL-17A production in mice. Targeting these receptors in EBV positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.
IMPORTANCE The Epstein-Barr virus is a pathogen that causes persistent infection with potential consistent viral DNA shedding. The enhancement of production of pro-inflammatory cytokines by viral DNA itself may contribute to autoimmune disease development or exacerbation. In this project we identified that endosomal Toll-like receptors are involved in triggering pro-inflammatory mediators in response to viral DNA. Pathways and receptors involved may serve as future therapeutic targets in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.
Vesicular Stomatitis Indiana Virus (VSIV), formerly known as Vesicular Stomatitis Virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons. Interferons induce an antiviral state by stimulating the expression of hundreds of interferon stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis, but helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley Fever Virus and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.
IMPORTANCE Vesicular Stomatitis Indiana Virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here we show that TRIM69 is an antiviral defence that can potently and specifically block VSIV infection.
Non-segmented negative-strand RNA viruses including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane-bound and serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggest that Mononegavirales broadly have evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells.
Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur and globally ~100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WDR5 enhances MeV virus growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies; that these structures display properties characteristic of phase-separated liquid organelles; and, that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.
Influenza A virus (IAV) non-structural protein 1 (NS1), a potent antagonist of host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of ED with respect to RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 (H6N6-NS1(E71)), and the LR-deletion mutant H6N6-NS1(80-84/E71) mimicking the major group H5N1-NS1, exhibit llsquo;openrrsquo;, llsquo;semi-openrrsquo;, and llsquo;closedrrsquo; conformations, respectively, suggesting that NS1 exhibits strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an llsquo;openrrsquo; conformation similar to the minor group of H5N1-NS1 (H5N1-NS1(G71)). We also show that H6N6-NS1(80-84/E71) under a different crystallization condition and H6N6-NS1(80-84/G71) also exhibit llsquo;openrrsquo; conformations, suggesting NS1 can adopt an llsquo;openrrsquo; conformation irrespective of E or G at the 71st position. Our single-molecule FRET analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in llsquo;openrrsquo; conformation. Further, our co-immunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinity. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection.
IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, non-structural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent llsquo;openrrsquo;, llsquo;semi-openrrsquo;, and llsquo;closedrrsquo; conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt llsquo;openrrsquo; conformation. We further show that NS1 from different strains primarily exists in llsquo;openrrsquo; conformation in solution and binds to cellular proteins with similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.
Long-acting antiretrovirals could provide a useful alternative to daily oral therapy for HIV-1 infected individuals. Building on a bi-specific molecule with adnectins targeting CD4 and gp41, a potential long-acting biologic, GSK3732394, was developed with three independent and synergistic modes of HIV entry inhibition that potentially could be self-administered as a long-acting subcutaneous injection. Starting with the bi-specific inhibitor, an alpha-helical peptide inhibitor was optimized as a linked molecule to the anti-gp41 adnectin, with each separate inhibitor exhibiting at least single digit nanomolar (or lower) potency and a broad spectrum. Combination of the two adnectins and peptide activities into a single molecule was shown to have synergistic advantages in potency, resistance barrier and in the ability to inhibit HIV-1 infections at low levels of CD4 receptor occupancy, showing that GSK3732394 can work in trans on a CD4+ T cell. Addition of a human serum albumin molecule prolongs the half-life in a human CD4 transgenic mouse, suggesting that it may have potential as a long acting agent. To show that, GSK3732394 was highly effective in a humanized mouse model of infection. GSK3732394 is currently in human studies.
IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1 infected patients is through the development of long acting biologic inhibitors. Building on a bi-specific inhibitor approach targeting CD4 and gp41, a tri-specific molecule was generated with three distinct antiviral activities. The linkage of these three biologic inhibitors creates synergy that offer a series of advantages to the molecule. The addition of human serum albumin to the tri-specific inhibitor could allow it to function as a long acting self-administered treatment for patients with HIV infection. This molecule is currently in early clinical trials.
The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent amongst the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a non-enveloped arbovirus, causing haemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical and microscopic techniques, we examine the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilising BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.
IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organisation; prominent members include heat shock protein 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host-factors is of primary importance for the development of intervention strategies. Our data identify and show that heat-shock protein 90, but not heat-shock protein 70, stabilizes Bluetongue virus proteins, safeguarding them from proteasomal degradation.
Replication of many (+)RNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. A non-structural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here we systematically investigated the conserved elements of GBF1 to understand what determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that Arf activating property, but not the primary structure of the Sec7 domain is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, dependent not only on direct interaction of the protein with the viral protein 3A, but also on determinants located in the non-catalytic C-terminal domains of GBF1. Such double targeting mechanism explains the previous observations of remarkable tolerance of different levels of GBF1-3A interaction by the virus, and likely constitutes an important element of resilience of viral replication.
Importance. Enteroviruses are a vast group of viruses associated with diverse human diseases but only two of them could be controlled with vaccines, and effective anti-viral therapeutics are lacking. Here we investigated in details the contribution of a cellular protein GBF1 in the replication of poliovirus, a representative enterovirus. GBF1 supports functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our resuts demonstrate that the virus requires a limited subset of the normal GBF1 functions, and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad spectrum anti-viral therapeutics.
Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates protein synthesis to meet cellular anabolic demands. In nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eIF4F disassembly and diminished global protein synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for synthesis of the RTA lytic switch protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 is not required for genome replication, late gene expression or release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together these findings suggest that mTORC1 is dispensable for viral protein synthesis and viral control of autophagy during lytic infection, and that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.
All viruses require host cell machinery to synthesize viral proteins. A host cell protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. In nutrient-rich conditions, mTORC1 is active and promotes protein synthesis to meet cellular anabolic demands. In nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) stimulates mTORC1 activity and utilizes host machinery to synthesize viral proteins. However, we discovered that mTORC1 activity was largely dispensable for viral protein synthesis, genome replication and release of infectious progeny. Likewise, during lytic replication, mTORC1 was no longer able to control autophagy. These findings suggest that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.
Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm in transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that hnRNP A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNA1 maps within the amino terminus (aa 19-40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. Furthermore, we uncovered that the N-terminal 21 amino acids (aa 19-40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41-60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, this study provides mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.
IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remains largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified a NES-containing nucleocytoplasmic shuttling domain (aa 19-40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulates nucleocytoplasmic shuttling of the ARV p17 protein.
BLT (bone marrow, liver, thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with HIV-1. We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein utilizing a rapid prime-boost protocol of PLGA microparticles and a replication-defective HSV recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide IFN-+ T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1 infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although pre-challenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these post-challenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5 log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including IL-12p70 and MIP-1aalpha;, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization, however additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy.
IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small animal model for pre-clinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable pre-challenge responses, it did increase the magnitude and kinetics of post-challenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.
Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytia formation in cultured human cells. Based on sequence alignment, NSP1-1 from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multi-cycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.
IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.
Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surface of infected cells. Recent evidence identified HLA-E, a non-classical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8+ T-cell and natural killer (NK)-cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1nndash;infected cells. Here, we examined HLA-E surface levels following infection of CD4+ T cells with primary HIV-1 strains and observed that a subset downregulates HLA-E. Two primary strains of HIV-1 inducing the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in T-cell lines as well as tail-swap experiments exchanging the cytoplasmic tail of HLA-A2 with HLA-E demonstrated that Nef modulated HLA-E surface levels in a cytoplasmic tail-dependent manner. Furthermore, infection of primary CD4+ T cells with HIV-1 mutants showed that lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, this study demonstrates for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4+ T cells, potentially rendering them less vulnerable to CD8+ T-cell recognition but at increased risk of NKG2A+ NK-cell killing.
IMPORTANCE For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that, thereby, HIV-1 infection maintains inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell-line adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in a significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as important modulator of CD8+ T-cell and NKG2A+ NK-cell function, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.
Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.
IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level, and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which a RNA virus interrupts host DNA replication. Lastly, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.
Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cells interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection, MOI) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOI, resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOI. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.
IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics by seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for the defense against the virus, and therefore it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells function as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.
Cytomegalovirus (CMV) is a ubiquitous bbeta;-herpesvirus that infects many different cell types. Human (H)CMV has been found in several solid tumors and it has been hypothesized that it may promote cellular transformation or exacerbate tumor growth. Paradoxically, in some experimental situations, murine (M)CMV infection delays tumor growth. We previously showed that wild-type MCMV delayed the growth of poorly immunogenic B16 melanomas via an undefined mechanism. Here we show that MCMV delayed the growth of these immunologically "cold" tumors by recruiting and modulating tumor-associated macrophages. Depletion of monocytic phagocytes with clodronate completely prevented MCMV from delaying tumor growth. Mechanistically, our data suggest that MCMV recruits new macrophages to the tumor via the virus-encoded chemokine MCK2, and viruses lacking this chemokine were unable to delay tumor growth. Moreover, MCMV infection of macrophages drove them toward an M1-like state. Importantly, adaptive immune responses were also necessary for MCMV to delay tumor growth as the effect was substantially blunted in Rag-deficient animals. However, viral spread was not needed and a spread-defective MCMV strain was equally effective. In most mice, the anti-tumor effect of MCMV was transient. Although the recruited macrophages persisted, tumor regrowth correlated with a loss of viral activity in the tumor. However, an additional round of MCMV infection further delayed tumor growth, suggesting that tumor growth delay was dependent on active viral infection. Together, our results suggest that MCMV infection delayed the growth of an immunologically "cold" tumor by recruiting and modulating macrophages in order to promote anti-tumor immune responses.
Importance Cytomegalovirus (CMV) is an exciting new platform for vaccines and cancer therapy. Although CMV may delay tumor growth in some settings, there is also evidence that CMV may promote cancer development and progression. Thus, defining the impact of CMV on tumors is critical. Using a mouse model of melanoma, we previously found that murine (M)CMV delayed tumor growth and activated tumor-specific immunity, although the mechanism was unclear. We now show that MCMV delayed tumor growth through a mechanism that required monocytic phagocytes and a viral chemokine that recruited macrophages to the tumor. Furthermore, MCMV infection altered the functional state of macrophages. Although the effects of MCMV on tumor growth were transient, we found that repeated MCMV injections sustained the anti-tumor effect suggesting that active viral infection was needed. Thus, MCMV altered tumor growth by actively recruiting macrophages to the tumor where they were modulated to promote anti-tumor immunity.
Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activity following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in the wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells but the RNA-containing CPMV particles were uniquely able to boost the populations of potent antigen presenting cells, such as tumor infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses.
The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical properties and immunostimulatory properties of wild type cowpea mosaic virus (CPMV, RNA-containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents.
Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells with FDA approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate nucleic acid packaging, transcription, and translation, but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential pro-viral role for polyamines using a forward screen. Passaging CVB3 in polyamine depleted cells generated a mutation in capsid protein VP3 at residue 234. We show this mutation confers resistance to polyamine depletion. Through attachment assays, we demonstrate that polyamine depletion limits CVB3 attachment to susceptible cells, which is rescued by incubating virus with polyamines. Further, the capsid mutant rescues this inhibition in polyamine depleted cells. More divergent viruses also exhibited reduced attachment to polyamine depleted cells, suggesting that polyamines may facilitate attachment of diverse RNA viruses. These studies inform additional mechanisms of action for polyamine-depleting pharmaceuticals with implications for potential antiviral therapies.
IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease. These viruses rely on polyamines, small positively-charged molecules, for robust replication, and polyamine depletion limits infection in vitro and in vivo. The mechanisms by which polyamines enhance enteroviral replication are unknown. Here, we describe how Coxsackievirus B3 (CVB3) utilizes polyamines to attach to susceptible cells and initiate infection. Using a forward genetic screen, we identified a mutation in a receptor-binding amino acid that promotes infection of polyamine-depleted cells. These data suggest that inhibiting polyamine biosynthesis pharmacologically may combat virus infection by preventing virus attachment to susceptible cells.
The rotavirus polymerase, VP1, mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein, VP2. Transcription [positive-strand (+) RNA synthesis] by VP1 occurs within double-layered particles (DLPs), while genome replication [double-stranded (ds) RNA synthesis] by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally-competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral fivefold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264-267, 547-550, 614-620, 968-980, and 1022-1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 in regard to polymerase activity. We engineered 19 recombinant (r) VP1 proteins that contained single- or multi-point alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their non-mutated contact sites. These results move towards a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation.
Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase, VP1, mediates all stages of viral RNA synthesis, and it requires the core shell protein, VP2, for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions, and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance an understanding of a key step of the rotavirus replication cycle.
Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-Burkitt lymphoma cell line and confirmed upregulation of all three branches of the UPR and activation of EBV lytic viral gene expression. While immediate early, early and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. Clofoctol in combination with a PERK inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic upregulation are not limited to lymphoid tumor cell lines but also occur in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but doesn't lead to production of infectious virions may have particular value for lytic induction strategies in the clinical setting.
IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent i.e. most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic clofoctol as an activator of EBV lytic viral RNA and protein expression that does not lead to virion production.
Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS), nasopharyngeal carcinoma (NPC43) and B cells (M81) led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit, CK2bbeta;, suggesting that CK2bbeta; might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2bbeta;, but not the CK2aalpha; catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 associated with Zp in reporter assays and this was increased by additional CK2bbeta;. Together the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2bbeta;-mediated interaction.
IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor, c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.
Globoside (Gb4) is considered the primary receptor of parvovirus B19 (B19V), however, its expression does not correlate well with attachment and restricted tropism of the virus. The N-terminal of VP1 (VP1u) of B19V interacts with as-yet-unknown receptor required for virus internalization. In contrast to Gb4, the VP1u cognate receptor is expressed exclusively in cells that B19V can internalize. With the aim to clarify the role of Gb4 as B19V receptor, we knocked out the gene B3GalNT1 encoding for the enzyme globoside synthase in UT7/Epo cells. Consequently, B3GalNT1 transcripts and Gb4 became undetectable in the knockout (KO) cells without affecting cell viability and proliferation. Unexpectedly, virus attachment, internalization, and nuclear targeting were not disturbed in the KO cells. However, NS1 transcription failed and consequently, genome replication and capsid protein expression were abrogated. The block could be circumvented by transfection with a B19V infectious clone, indicating that Gb4 is not required after the generation of viral dsDNA with resolved inverted terminal repeats. While in wild-type cells (WT), occupation of the VP1u cognate receptor with recombinant VP1u disturbed virus binding and blocked the infection, antibodies against Gb4 had no significant effect. In a mixed population of WT and KO cells, B19V selectively infected WT cells. This study demonstrates that Gb4 does not have the expected receptor function as it is dispensable for virus entry, however, is essential for productive infection, explaining the resistance of the rare individuals lacking Gb4 to B19V infection.
Vaccination is widely used to generate protective immunity to influenza. CD4+ T cells contribute in diverse ways to protective immunity, most notably for provision of help for production of neutralizing antibodies. Several recent reports have suggested that influenza infection elicits CD4+ T cells whose specificity only partially overlaps with those elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4+ T cell repertoire, we have evaluated protein vaccine- vs. infection-generated CD4+ T cell epitopes. Our studies revealed that all the infection-elicited CD4+ T cell epitope specificities are also elicited by protein vaccination, although immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus, and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4+ memory T cells are recalled and boosted after infection and provide early help to accelerate HA-specific antibody responses. The early CD4+ T cell response and HA-specific antibody production is associated with lowered viral titers during the infection challenge. Our data lend confidence in current protein-based vaccines ability to elicit influenza-specific CD4+ T cells that can potentiate protective immunity upon influenza virus infection.
Importance. Most current and new influenza vaccine candidates consist of single or combinations of influenza virus proteins. For these vaccines to elicit CD4+ T cells that can be recalled after infection, the peptides epitopes should be shared between the two modes of confrontation. There have recently been questions raised regarding the relatedness of epitope selection by influenza infection and protein vaccination. However, the studies reported here show that protein-based vaccines overlap with those elicited by infection and that CD4+ T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection.
The RV144 HIV-1 vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hotspot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA-SHIV and boosting with modified vaccinia Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile. Two gp140 booster immunizations induced very high levels (~2mg/ml) of gp140 binding antibodies in serum with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3 and gp41 immunodominant (gp41 ID) proteins. However, the vaccine induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/MVA vaccination do not significantly boost heterologous V1V2 and V2HS responses, and fail to enhance protection against heterologous SHIV challenge.
IMPORTANCE - HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually. Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already well-established DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune correlates for protection in a moderately successful RV144 HIV vaccine trial in humans, and highlight the need for the development of improved HIV envelope immunogens.
Comparative examination of viral and host homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called VRKs (vaccinia related kinases). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1 as compared to other DNA viruses. Employing loss of function analysis, we demonstrate that vaccinia dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining B1 and VRK2 co-precipitation of B12. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating, but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analysis of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling co-regulation and support a model in which these enzymes modulate B12 in a phosphorylation dependent manner.
Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious, mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia B12 via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog, VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.
A relatively stable and flexible capsid is critical to viral life cycle. However, the capsid dynamics and trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation of PCV2 virions by genome labelling and the use of three monoclonal antibodies against native capsid of PCV2. Genome labelling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65ddeg;C causing all antibodies binding incompetence, viral genome was released without capsid disassembly. Antibodies binding experiments with PCV2 virions showed that residues 186 to 192 was concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment, then exposed in PK-15 cytosol and 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking was not significantly affected in monocytic 3D4/31 cells treated with nocodazole by microtubule depolymerization. These findings demonstrated that nuclear-targeting of viral capsids involved conformational changes, PCV2 genome was released from assembled capsid, the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.
IMPORTANCE Circovirus is the smallest virus known to be autonomously replicable. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of PCV2. Here, we report the release of the PCV2 genome from assembled capsid, the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious diseases affecting the global pig industry. Previous studies from our and other groups showed that cholesterol 25-hydroxylase (CH25H), a multi-transmembrane endoplasmic reticulum-associated enzyme, catalyzes the production of 25-hydroxycholesterol (25HC) and inhibits PRRS virus (PRRSV) replication. However, PRRSV infection also actively decreases porcine CH25H (pCH25H) expression through unidentified mechanism(s). In this study, we found that the ubiquitin-proteasome pathway plays a major role in pCH25H degradation during PRRSV infection, and that the PRRSV-encoded envelope (E) protein interacts with pCH25H. PRRSV E protein degraded pCH25H via ubiquitination, and the ubiquitination site was at pCH25H Lys28. Interestingly, PRRSV E protein appeared to specifically degrade pCH25H but not human CH25H, likely because of a Lys28Arg substitution in the human orthologue. As expected, ubiquitin-mediated degradation by E protein attenuated the antiviral effect of pCH25H by downregulating 25HC production. In addition, we found that knockdown of pCH25H decreased E protein-induced inflammatory cytokine expression and that pCH25H overexpression had the opposite effect. These findings suggested that regulation of pCH25H expression was associated with E protein-induced inflammatory responses. Taken together, our results and those of previous studies of the anti-PRRSV effects of CH25H highlight the complex interplay between PRRSV and pCH25H.
Cholesterol 25-hydroxylase (CH25H) has received significant attention due to its broad antiviral activity, which it mediates by catalyzing the production of 25-hydroxycholesterol (25HC). Most studies have focused on the antiviral mechanisms of CH25H; however, whether viruses also actively regulate CH25H expression has not yet been reported. Previous studies demonstrated that porcine CH25H (pCH25H) inhibits PRRSV replication not only via production of 25HC, but also by ubiquitination and degradation of viral nonstructural protein 1aalpha;. In this study, we expanded on previous work and found that PRRSV actively degrades pCH25H through the ubiquitin-proteasome pathway. PRRSV envelope protein, a viral structural protein, is involved in this process. This study reveals a novel interaction mechanism between virus and host during PRRSV infection.
Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTV viruses have only been reported in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within Open Reading Frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (llt;100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread.
Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade.
Endogenous retroviruses (ERV) are found throughout vertebrate genomes and failure to silence their activation can have deleterious consequences on the host. Mutation and subsequent disruption of ERV loci is therefore an indispensable component of the cell-intrinsic defenses that maintain the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G) can potently restrict retrotransposition; yet in vivo data demonstrating such activity is lacking, since no replication competent human ERV have been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that ectopic expression of hA3G can prevent the emergence of replication-competent, infectious ERV in Tlr7-/- mice. Mice encode one copy of Apobec3 in their genome. ERV reactivation in Tlr7-/- mice was comparable in the presence and absence of Apobec3. In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7-/- background. No ERV RNA was detected in the plasma of hA3G+Apobec3-/-Tlr7-/- mice, and infectious ERV virions could not be amplified through co-culture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo, and suggest that expansion of the APOBEC3 locus in primates may have helped to provide for the continued restraint of ERV in the human genome.
Although APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) has been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.
The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), III (D). Alanine substitution of the motif I residues (AA-E-D, four nucleotide substitutions) in murine hepatitis virus (MHV) and SARS-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2 or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to MHV-ExoN-AA. In contrast, a low passage (P10) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multi-protein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.
Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside of ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.
Superinfection exclusion (SIE) or "cross protection" phenomena have been documented for plant viruses for nearly a century and are widespread among taxonomically diverse viruses, but little information is available about SIE of plant negative-strand RNA viruses. Here we demonstrate that SIE by sonchus yellow net nucleorhabdovirus virus (SYNV) is mediated by the viral matrix (M) protein, a multi-functional protein involved in transcription regulation, virion assembly and virus budding. We show that fluorescent protein-tagged SYNV variants display mutual exclusion/cross protection in Nicotiana benthamiana plants. Transient expression of the SYNV M protein, but not other viral proteins, interfered with SYNV local infections. In addition, SYNV M deletion mutants failed to exclude superinfection by wild-type SYNV. An SYNV minireplicon reporter gene expression assay showed that the M protein inhibited viral transcription. However, M protein mutants with weakened nuclear localization signals (NLS) and deficient nuclear interactions with the SYNV nucleocapsid protein were unable to suppress transcription. Moreover, SYNV with M NLS mutations exhibited compromised SIE against wild-type SYNV. From these data, we propose that M protein accumulating in nuclei with primary SYNV infections either coils or prevents uncoiling of nucleocapsids released by the superinfecting SYNV virions and suppresses transcription of superinfecting genomes, thereby preventing superinfection. Our model suggests that the rhabdovirus M protein regulates the transition from replication to virion assembly and renders the infected cells non-permissive for secondary infections.
IMPORTANCE Superinfection exclusion (SIE) is a widespread phenomenon in which an established virus infection prevents re-infection by closely related viruses. Understanding the mechanisms governing SIE will not only advance our basic knowledge of virus infection cycles, but may also lead to improved design of anti-viral measures. Despite its significance, our knowledge about viral SIE determinants and their modes of actions remain limited. In this study, we show that sonchus yellow net virus (SYNV) SIE is mediated by the viral matrix (M) protein. During primary infections, accumulation of M protein in infected nuclei results in coiling of genomic nucleocapsids and suppression of viral transcription. Consequently, nucleocapsids released by potential superinfectors are sequestered and are unable to initiate new infections. Our data suggest that SYNV SIE is caused by M protein-mediated transition from replication to virion assembly and that this process prevents secondary infections.
Proteoglycans function not only as structural components of the extracellular compartment but also as regulators of various cellular events, including cell migration, inflammation, and infection. Many microbial pathogens utilize proteoglycans to facilitate adhesion and invasion into host cells. Here, we report a secreted form of a novel heparan sulfate proteoglycan that functions against virus infection. The expression of SPOCK2/testican2 was significantly induced in virus-infected lungs or in IFN-treated alveolar lung epithelial cells. Overexpression from a SPOCK2 expression plasmid alone or the treatment of cells with recombinant SPOCK2 protein efficiently blocked influenza virus infection at the step of viral attachment to the host cell and entry. Moreover, mice treated with purified SPOCK2 were protected against virus infection. Sialylated glycans and heparan sulfate chains covalently attached to the SPOCK2 core protein were critical for its antiviral activity. Neuraminidase (NA) of influenza virus cleaves the sialylated moiety of SPOCK2, thereby blocking its binding to the virus. Our data suggest that IFN-induced SPOCK2 functions as a decoy receptor to bind and block influenza virus infection, thereby restricting entry of the infecting virus into neighboring cells.
IMPORTANCE Here, we report a novel proteoglycan protein, testican2/SPOCK2, that prevents influenza virus infection. Testican/SPOCK2 is a complex type of secreted proteoglycan with heparan sulfate GAG chains attached to the core protein. SPOCK2 expression is induced upon virus infection or by interferons and it is secreted to an extracellular compartment, where it acts directly to block virus-cell attachment and entry. Treatment with purified testican/SPOCK2 protein can efficiently block influenza virus infection in vitro and in vivo. We also identified the heparan sulfate moiety as a key regulatory module for this inhibitory effect. Based on its mode of action (cell-attachment/entry blocker) and site of action (extracellular compartment), we propose testican/SPOCK2 as a potential antiviral agent that can efficiently control influenza virus infection.
The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wildtype, replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by over-expression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not MLV infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication by altering the cellular milieu through targeting factors including TNPO3.
HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study we identified the interferon-inducible miR-128, as a novel antiviral mediator, which suppresses the expression of the host gene TNPO3 known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.
Epstein-Barr virus is associated with several human malignancies including nasopharyngeal carcinoma, gastric cancer and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (OriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr Nuclear Antigen-1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here, we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half site. This structure shows that the DNA is smoothly bent allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised OriP-containing plasmid replication in cells and impaired recruitment of the MCM3 complex to the OriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the OriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.
Epstein-Barr Virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) with the viral origin of plasmid replication OriP. The Dyad Symmetry (DS) element in OriP is the essential minimal replicator of OriP. Here, we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA-binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.