Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling

Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Journal of Virology (Impact Factor: 4.44). 12/2007; 81(21):11809-16. DOI: 10.1128/JVI.00791-07
Source: PubMed


West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis.
Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing
the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350,
2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls
IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of
infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient
to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in
the inhibition of the IFN signaling pathway during WNV infections.

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    • "Mutation studies in the NS4B protein have suggested that it is associated with both viral replication and evasion of host immunity [11] [12] [13]. The NS4B P38 residue is conserved in the majority of mosquito-and tick-borne flaviviruses. "
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    ABSTRACT: Prior work shows that an attenuated West Nile virus (WNV), the nonstructural (NS)4B-P38G mutant infection in mice induced strong immune responses and protected host from subsequent lethal wild-type WNV infection. Here, we investigated NS4B-P38G mutant infection in myeloid differentiation factor 88-deficient (MyD88(-/-)) and Toll-like receptor 7-deficient (TLR7(-/-)) mice and found they had enhanced susceptibility compared to wild-type mice. Both groups had lower WNV-specific IgM response and reduced effector T cell functions. Dendritic cells (DCs) also exhibited a reduced maturation and impaired antigen-presenting functions compared to wild-type DCs. Moreover, infection with NS4B-P38G mutant in TLR7(-/-) and MyD88(-/-) mice provided full and partial protection respectively from subsequent challenge with lethal wild-type WNV. There were reduced T cell responses in MyD88(-/-) and interleukin-1 receptor deficient (IL-1R(-/-)) mice during secondary challenge with wild-type WNV. In contrast, TLR7(-/-) mice displayed normal T cell functions. Collectively, these results suggest that TLR7-dependent MyD88 signaling is required for T cell priming during NS4B-P38G mutant infection, whereas the TLR7-independent MyD88 signaling pathways are involved in memory T cell development, which may contribute to host protection during secondary challenge with wild-type WNV.
    Full-text · Article · Jul 2013 · Vaccine
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    • "Overall, the structural proteins are involved in virus binding and penetration of host cells [19], while the non-structural proteins are involved in the replicative cycle [20] and induce immunological evasion mainly through the inhibition of type I interferon signaling [21,22]. Still, it has been suggested that other unidentified factors could play a role in the pathogenesis of WNV neuroinvasive disease [23]. Melian et al. demonstrated that the NS1’ protein, a known variant of the canonical NS1 protein, results from a ribosomal frame shifting process [24]. "
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    ABSTRACT: Background West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. Results We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. Conclusions N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.
    Full-text · Article · Nov 2012 · Virology Journal
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    • "IS-98-ST1 is suitable for the study of viral determinants of WNV virulence, as well as host factors involved in viral pathogenicity [21], [22], [23], [24], [25]. To date, a few molecular clones derived from North American, African and Australian strains of WNV are available for the study of viral neuropathogenicity [1], [3], [9], [26], [27], [28]. We report here that WNV strain IS-98-ST1 recovered from an infectious cDNA clone reproduces the pathobiological properties of the parental strain, and is thus well-suited for studying the virulence of WNV isolates from Europe/West Asia. "
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    ABSTRACT: Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.
    Full-text · Article · Oct 2012 · PLoS ONE
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