- Hepatitis B spliced protein (HBSP) suppresses Fas-mediated hepatocyte apoptosis via activation of PI3K/Akt signaling. [Journal Article]
- JVJ Virol 2018 Sep 12
- Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis, however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-...
Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis, however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-mediated apoptosis is thought as one of the major promotors for hepatitis B virus (HBV) chronicity and malignancy. The purpose of this study was to investigate whether HBSP could protect hepatocytes against Fas-initiated apoptosis. We showed here that HBSP mediated resistance of hepatoma cells or primary human hepatocytes (PHH) to agonistic anti-Fas antibody (CH11)- or FasL-induced apoptosis. Under Fas signaling stimulation, expression of HBSP inhibited Fas aggregation and prevented recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 (or FADD like interleukin 1 β-converting enzyme, FLICE) into the death-inducing signaling complex (DISC), while increasing recruitment of cellular FLICE-inhibitory protein L (FLIPL) into the DISC. Those effects may be mediated through activation of PI3K/AKT pathway as evidenced by increased cellular PIP3 content and PI3K activity, and enhanced phosphorylation of mTORC2 and PDPK1 as well as Akt itself. Confirmedly, inhibition of PI3K by LY294002 reversed the effect of HBSP on Fas aggregation, FLIPL expression and cellular apoptosis. These results indicate that HBSP functions to prevent hepatocytes from Fas-induced apoptosis by enhancing PI3K/Akt activity, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.Importance: Our study revealed a previously unappreciated role of HBSP in Fas-mediated apoptosis. The anti-apoptotic activity of HBSP is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with hepatoma carcinoma may down-regulate apoptosis of hepatocytes through enhanced HBSP expression. Our study also found that AKT is centrally involved in Fas-induced hepatocyte apoptosis and revealed interventions directed at inhibiting the activation or functional activity of AKT may be of therapeutic value in this process.
- Type I interferon signaling prevents hepatitis B virus-specific T cell responses by reducing antigen expression. [Journal Article]
- JVJ Virol 2018 Sep 12
- Robust virus-specific CD8+ T cell responses are required for the clearance of hepatitis B virus (HBV). However, the factors that determine the magnitude of HBV-specific CD8+ T cell responses are poor...
Robust virus-specific CD8+ T cell responses are required for the clearance of hepatitis B virus (HBV). However, the factors that determine the magnitude of HBV-specific CD8+ T cell responses are poorly understood. To examine the impact of genetic variations of HBV on HBV-specific CD8+ T cell responses, we introduced three HBV clones (Aa, C22, D60) that express varying amounts of HBV antigens into the livers of C57BL/6 (B6) mice (H-2b) and B10.D2 mice (H-2d). In B6 mice, clone C22 barely induced HBV-specific CD8+ T cell responses and persisted the longest, while clone D60 elicited strong HBV-specific CD8+ T cell responses and was rapidly cleared. These differences between HBV clones largely diminished in B10.D2 mice (H-2d). Interestingly, the magnitude of HBV-specific CD8+ T cell responses in B6 mice was associated with the HB core antigen expression level during the early phase of HBV transduction. Surprisingly, robust HBV-specific CD8+ T cell responses to clone C22 were induced in interferon-α/β receptor-deficient (IFN-αβR-/-) mice (H-2b). The induction of HBV-specific CD8+ T cell responses to C22 in IFN-αβR-/- mice reflects enhanced HBV antigen expression because the suppression of antigen expression by HBV-specific siRNA attenuated HBV-specific T cell responses in IFN-αβR-/- mice and prolonged HBV expression. Collectively, these results suggest that HBV genetic variation and type I interferon signaling determine the magnitude of HBV-specific CD8+ T cell responses by regulating the initial antigen expression levels.IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic infection, and approximately 240 million people are chronically infected with HBV worldwide. It is generally believed that virus-specific CD8+ T cell responses are required for the clearance of HBV. However, the relative contribution of genetic variation and innate immune responses to the induction of HBV-specific CD8+ T cell responses are not fully understood. In this study, we discovered that different clearance rate between HBV clones after hydrodynamic transduction was associated with the magnitude of HBV-specific CD8+ T cell responses and initial HB core antigen expression. Surprisingly, type I interferon signaling negatively regulated HBV-specific CD8+ T cell responses by reducing early HBV antigen expression. These results show that the magnitude of HBV-specific CD8+ T cell response is primarily regulated by the initial antigen expression level.
- Recombinant Infectious Bronchitis Viruses expressing chimaeric spike glycoproteins induce partial protective immunity against homologous challenge despite limited replication in vivo. [Journal Article]
- JVJ Virol 2018 Sep 12
- Vaccination regimes against Infectious bronchitis virus, which are based on a single virus serotype, often induce insufficient levels of cross-protection against serotypes and two or more antigenical...
Vaccination regimes against Infectious bronchitis virus, which are based on a single virus serotype, often induce insufficient levels of cross-protection against serotypes and two or more antigenically diverse vaccines are used in attempt to provide broader protection. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, are associated with poor cross-protection. Here, homologous vaccination trials with recombinant IBVs, based on the apathogenic strain, BeauR, were conducted to elucidate the role of S1 in protection. A single vaccination of SPF-chickens with rIBV expressing S1 of virulent strains M41 or QX, BeauR-M41(S1) and BeauR-QX(S1), gave incomplete protection against homologous challenge, based on ciliary activity and clinical signs. There could be conformational issues with the spike if heterologous S1 and S2 are linked, suggesting a homologous S2 might be essential. To address this, a homologous vaccination-challenge trial incorporating rIBVs expressing full spike from M41, BeauR-M41(S), and S2 subunit from M41, BeauR-M41(S2) was conducted. All chimaeric viruses grew to similar titres in vitro, induced virus-specific partial protective immunity, evident by cellular infiltrations, reductions in viral RNA load in the trachea and conjunctiva and higher serum anti-IBV titres. Collectively, these show that vaccination with rIBVs primed the birds for challenge but the viruses were cleared rapidly from the mucosal tissues in the head. Chimaeric S1 and S2 viruses did not protect as effectively as BeauR-M41(S) based on ciliary activity and clinical signs. Booster vaccinations and a rIBV with improved in vivo replication may improve the levels of protection.IMPORTANCE Infectious bronchitis virus causes an acute, highly contagious respiratory disease, responsible for significant economic losses to the poultry industry. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, have been associated with poor cross-protection. Available vaccines give poor cross-protection and rationally designed live attenuated vaccines, based on apathogenic BeauR, could address these. Here, to determine the role of S1 in protection, a series of homologous vaccination trials with rIBVs were conducted. Single vaccinations with chimaeric rIBVs induced virus-specific partial protective immunity, characterised by reduction in viral load and serum antibody titres. However, BeauR-M41(S) was the only vaccination to improve the level of protection against clinical signs and the loss of tracheal ciliary activity. Growth characteristics show all of the rIBVs replicated in vitro to similar levels. Booster vaccinations and a rIBV with improved in vivo replication may improve the levels of protection.
- HOIL1 is essential for the induction of type I and III interferons by MDA5 and regulates persistent murine norovirus infection. [Journal Article]
- JVJ Virol 2018 Sep 12
- The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase-1 (HOIL1), HOIL-1-interacting protein (HOIP) and SHANK-associated RH-domain-interacting protein (S...
The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase-1 (HOIL1), HOIL-1-interacting protein (HOIP) and SHANK-associated RH-domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1- or HOIP-deficiency is associated with an immune disorder involving auto-inflammation, immunodeficiency and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor, RIG-I. Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, MDA5, is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I:C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required specifically for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus (MNoV) infection, and defective regulation of MNoV persistence by the microbiome as also observed previously for IFN-λ receptor-, STAT1- and IRF3-deficient mice. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world, but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small animal model to study norovirus biology and immune responses. Interferons are critical mediators of anti-viral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. The LUBAC complex, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.
- Distribution, diversity and evolution of endogenous retroviruses in perissodactyl genomes. [Journal Article]
- JVJ Virol 2018 Sep 12
- The evolution of mammalian genomes has been shaped by interactions with endogenous retroviruses (ERVs). In this study, we investigated the distribution and diversity of ERVs in the mammalian order Pe...
The evolution of mammalian genomes has been shaped by interactions with endogenous retroviruses (ERVs). In this study, we investigated the distribution and diversity of ERVs in the mammalian order Perissodactyla, with a view to understanding their impact on the evolution of modern equids (family Equidae). We characterize the major ERV lineages in the horse genome in terms of their genomic distribution, ancestral genome organization and time of activity. Our results show that subsequent to their ancestral divergence from rhinos and tapirs, equids acquired four novel ERV lineages. We show that two of these proliferated extensively in the lineage leading to modern horses, and one contains loci that are actively transcribed in specific tissues. In addition, we show that the white rhinoceros has resisted germline colonisation by retroviruses for over 54 million years - longer than any other extant mammalian species. The map of equine ERVs that we provide here will be of great utility to future studies aiming to investigate the potential functional roles of equine ERVs, and their impact on equine evolution.IMPORTANCE ERVs in the host genome are highly informative about the long-term interactions of retroviruses and hosts. They are also interesting because they have influenced the evolution of mammalian genomes in various ways. In this study, we derive a calibrated timeline describing the process through which ERV diversity has been generated in the equine germline. We determined the distribution and diversity of perissodactyl ERV lineages and inferred their retrotranspositional activity during evolution, thereby gaining insight into the long-term co-evolutionary history of retroviruses and mammals. Our study provides a platform for future investigations to identify equine ERV loci involved in physiological processes and/or pathological conditions.
- Human host-range restriction of the vaccinia virus C7/K1 double deletion mutant is mediated by an atypical mode of translation inhibition. [Journal Article]
- JVJ Virol 2018 Sep 12
- Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide siRNA screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host-ra...
Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide siRNA screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host-range restriction factor plus additional candidates including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knock-out (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80s and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained non-permissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other's vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host-range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L or G3 binding proteins, suggesting a novel mechanism of host restriction.
- Susceptibility to Neutralization by Broadly Neutralizing Antibodies Generally Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs. [Journal Article]
- JVJ Virol 2018 Sep 12
- Efforts to HIV cure are obstructed by reservoirs of latently infected CD4+ T-cells that can re-establish viremia. Broadly neutralizing HIV-specific antibodies (bNAbs), defined by unusually high neutr...
Efforts to HIV cure are obstructed by reservoirs of latently infected CD4+ T-cells that can re-establish viremia. Broadly neutralizing HIV-specific antibodies (bNAbs), defined by unusually high neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T-cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected-cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T-cells of ARV-treated HIV-infected individuals, using paired neutralization and infected-cell binding assays. Single antibody breadths ranged from 0-64% for neutralization (IC80≤10μg/ml) and 0-89% for binding, with two-antibody combinations (antibody combinations are theoretical/predicted) reaching 0-83% and 50-100%, respectively. Infected-cell binding correlated with virus neutralization for 10 out of 14 antibodies (e.g. 3BNC117, r=0.82, p<0.0001). Heterogeneity was observed, however, with a lack of significant correlations for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies; both by providing a direct assessment of intra- and inter-individual variability in neutralization and infected cell binding in a novel cohort, and by defining the relationships between these parameters for a panel of bNAbs.Importance Although anti-retroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their abilities to both prevent the infection of new cells (neutralization), and also to direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication, and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected-cell binding, we observed variation both between individuals, and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.
- The nucleolar protein LYAR facilitates ribonucleoprotein assembly of influenza A virus. [Journal Article]
- JVJ Virol 2018 Sep 12
- Influenza A viral ribonucleoprotein (vRNP) is responsible for transcription and replication of the viral genome in infected cells and depend on host factors for its functions. Identification of the h...
Influenza A viral ribonucleoprotein (vRNP) is responsible for transcription and replication of the viral genome in infected cells and depend on host factors for its functions. Identification of the host factors interacting with vRNP not only improves understanding of virus-host interactions, but also provides insights into novel mechanisms of viral pathogenicity and the development of new antiviral strategies. Here, we have identified eighty host factors that co-purified with vRNP using affinity purification followed by mass spectrometry. LYAR, a cell growth-regulating nucleolar protein, has been shown to be important for influenza A virus replication. During influenza A virus infection, LYAR expression is increased and it partly translocates from the nucleolus to nucleoplasm and cytoplasm. Furthermore, LYAR interacts with RNP subunits, resulting in enhancing viral RNP assembly, thereby facilitating viral RNA synthesis. Taken together, our studies identify a novel vRNP binding host partner important for influenza A virus replication, and further reveal the mechanism of LYAR regulating influenza A viral RNA synthesis by facilitating viral RNP assembly.IMPORTANCE Influenza A virus (IAV) must utilize the host cell machinery to replicate, but many of the mechanisms of the IAV-host interaction remain poorly understood. Improved understanding of interactions between host factors and vRNP not only increases our basic knowledge of the molecular mechanisms of virus replication and pathogenicity, but also provides insights into possible novel antiviral targets that are necessary due to the widespread emergence of drug-resistant IAV strains. Herein, we have identified LYAR, a cell growth-regulating nucleolar protein, which interacts with viral RNP components and is important for efficient replication of IAVs, and whose role in the IAV life cycle has never been reported. In addition, we further reveal the role of LYAR in viral RNA synthesis. Our results extend and improve the current knowledge on the mechanisms of IAV transcription and replication.
- A single point mutation in the rhinovirus 2B protein reduces the requirement for phosphatidylinositol 4-kinase class 3beta in viral replication. [Journal Article]
- JVJ Virol 2018 Sep 12
- Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C and 3A, which control trafficking and lipid composition of the re...
Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as the phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels, and drives lipid counter-current exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein (OSBP) 1. We identified a PI4K3b-inhibitor resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy-terminus, isoleucine 92 to threonine [I92T]. The mutation did not confer resistance to cholesterol sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid counter-currents. In presence of PI4K3b-inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92], but not wild-type infection. The knock-out of PI4K3b abolished the replication of both 2B[I92T] mutant and wild-type. Doxycyclin-inducible expression of PI4K3b in PI4K3b knock-out cells efficiently rescued the 2B[I92T] mutant, and less effectively wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane, and allows the virus to adapt to cells with low levels of PI4K3b, yet maintaining the PI4P/cholesterol counter-current for establishing Golgi-derived RV replication membranes.ImportanceHuman rhinoviruses (RVs) are the major cause of common cold worldwide. They cause asthmatic exacerbations and chronic obstructive pulmonary disease. Despite recent advances, the development of antivirals and vaccines has proven difficult due to the high number and variability of RV types. The identification of critical host factors and their interactions with viral proteins and membrane lipids for the establishment of viral replication is a basis for drug development strategies. Our findings here shed new light on the interactions between nonstructural viral membrane proteins and class III phosphatidylinositol 4 kinases from the host, and highlight the importance of phosphatidyl-inositol 4 phosphate for RV replication.
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- Epstein-Barr Virus miR-BART5-3p inhibits p53 expression. [Journal Article]
- JVJ Virol 2018 Sep 12
- Epstein-Barr virus (EBV) is the first human virus found to encode many microRNAs. It is etiologically linked to nasopharyngeal carcinoma and EBV-associated gastric carcinoma. During the latent infect...
Epstein-Barr virus (EBV) is the first human virus found to encode many microRNAs. It is etiologically linked to nasopharyngeal carcinoma and EBV-associated gastric carcinoma. During the latent infection period, there are only a few EBV proteins expressed, whereas EBV microRNAs, such as BART microRNAs, are highly expressed. However, how these BART miRNAs precisely regulate the tumor growth in nasopharyngeal carcinoma and gastric carcinoma remains obscure. Here, we report that up-regulation of EBV-miR-BART5-3p promotes the growth of nasopharyngeal carcinoma and gastric carcinoma cells. BART5-3p directly targets the tumor suppressor gene TP53 on its 3'-UTR and consequently down-regulates CDKN1A, BAX and FAS expression, leading to acceleration of the cell cycle progress and inhibition of cell apoptosis. BART5-3p contributes to the resistance to chemotherapeutic drugs and ionizing irradiation-induced p53 increase. Moreover, BART5-3p also facilitates degradation of p53 proteins. BART5-3p is the first EBV-microRNA to be identified to inhibiting p53 expression and function, which suggests a novel mechanism underlying the strategies employed by EBV to maintain latent infection and promote the development of EBV-associated carcinomas.IMPORTANCE EBV encodes 44 mature microRNAs, which have been proven to promote EBV-associated diseases by targeting host genes and self-viral genes. In EBV-associated carcinomas, the expression of viral protein is limited but the expression of BART microRNAs is extremely high, suggesting that they could be major factors in the contribution of EBV-associated tumorigenesis. p53 is a critical tumor suppressor. Unlike in most human solid tumors, TP53 mutations are rare in nasopharyngeal carcinoma and EBV associated gastric carcinoma tissues, suggesting a possibility that some EBV-encoded products suppress the functions of p53. This study provides the first evidence that a BART microRNA can suppress p53 expression by directly targeting its 3'-UTR. This study implies that EBV can use its BART microRNAs to modulate the expression of p53, thus maintaining its latency and contributing to tumorigenesis.