Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Mouse STAT2 restricts early dengue virus replication

Dengue virus encodes several interferon antagonists. Among these the NS5 protein binds STAT2, a necessary component of the type I interferon signaling pathway, and targets it for degradation. We now demonstrate that the ability of dengue NS5 to associate with and degrade STAT2 is species specific. Thus, NS5 is able to bind and degrade human STAT2, but not mouse STAT2. This difference was exploited to demonstrate, absent manipulation of the viral genome, that NS5-mediated IFN antagonism is essential for efficient virus replication. Moreover, we demonstrate that differences in NS5 mediated binding and degradation between human and mouse STAT2 maps to a region within the STAT2 coiled-coil domain. By using STAT2(-/-) mice, we also demonstrate that mouse STAT2 restricts early dengue virus replication in?vivo. These results suggest that overcoming this restriction through transgenic mouse technology may help in the development of a long-sought immune-competent mouse model of dengue virus infection.     

Influenza B virus NS1-truncated mutants: live-attenuated vaccine approach

Type B influenza viruses can cause substantial morbidity and mortality in the population, and vaccination remains by far the best means of protection against infections with these viruses. Here, we report the construction of mutant influenza B viruses for potential use as improved live-virus vaccine candidates. Employing reverse genetics, we altered the NS1 gene, which encodes a type I interferon (IFN) antagonist. The resulting NS1 mutant viruses induced IFN and, as a consequence, were found to be attenuated in vitro and in vivo. The absence of pathogenicity of the NS1 mutants in both BALB/c and C57BL/6 PKR(-/-) mice was confirmed. We also provide evidence that influenza B virus NS1 mutants induce a self-adjuvanted immune response and confer effective protection against challenge with both homologous and heterologous B virus strains in mice.     

Japanese Encephalitis Virus NS1′ Protein Antagonizes Interferon Beta Production

Japanese encephalitis virus(JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a52-amino acid C-terminal extension of NS1, is generated with a-1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected(rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus(pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type Ⅰ interferon(IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-b and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.     

Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.     

Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2alpha (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208-5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262-1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2alpha after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2alpha phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.     

Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling

Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.