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  • WNV a member of the genus Flavivirus within the

    2019-11-06

    WNV, a member of the genus Flavivirus within the family Flaviviridae, was first isolated in 1937 from a febrile woman in the West Nile region of Uganda (Brinton, 2002). Until 1999, WNV was mainly confined to Southern Europe, the Middle East, Africa, West and Central Asia, Indonesia and Australia. In 1999, WNV extended into the Western hemisphere where it has since spread rapidly. The majority of WNV infections in humans are asymptomatic. Flu-like symptoms are observed in ~20% and meningitis, encephalitis and/or paralysis occur in less than 1% of infected individuals (Brinton, 2002, Gubler et al., 2007). The WNV genome is a positive-sense, single-stranded RNA of ~11kb with a 5′ cap but no 3′ polyA tract. It encodes a single polyprotein that is co- and post-translationally cleaved to generate 3 structural proteins (E, prM and C) and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). The steps of the viral life cycle take place in the cytoplasm. WNV infection does not lead to shut-off of cellular protein synthesis. Viral RNA replication occurs in vesicles formed by invaginations of the ER membranes (Lindenbach et al., 2007). Nascent virions are assembled through the interaction of viral structural proteins associated with ER membranes with a newly synthesized viral RNA genome followed by Caspase-3/7 Inhibitor into the lumen of the ER. Virions are transported through the Golgi system to the cell surface (Brinton, 2002, Gubler et al., 2007). PKR has been reported to play a role in NF-κB signaling and the control of cell growth through induction of p53 (Garcia et al., 2006) and also to be involved in IFN, PDGF, TNF-α, p38, JNK, STAT1 and IL-1 signaling (Garcia et al., 2006). The involvement of PKR in these multiple cellular processes requires its phosphorylation (Garcia et al., 2006). A number of cellular inhibitors have been identified that can form stable heterocomplexes with PKR and interfere with a step of the PKR activation process: (1) dsRNA recognition (C114 and RPL18), (2) dimerization (p58ipk) or (3) autophosphorylation (Hsp70 and Hsp90) (Garcia et al., 2007). The catalytic subunit of protein phosphatase 1 alpha (PP1a) dephosphorylates PKR resulting in dimer disruption (Tan et al., 2002). The importance of PKR as a sentinel for the antiviral innate immune response is highlighted by the many reports indicating that most known viruses have evolved mechanisms for inhibiting PKR activity (Garcia et al., 2007). Viral components can either directly inhibit PKR activation or recruit cellular PKR inhibitors. Viral proteins, including Kaposi-sarcoma herpesvirus vIRF2 and LANA2, herpes simplex virus 1 (HSV-1) Us11, Epstein-Barr virus SM, vaccinia virus E3L and hepatitis C virus NS5A and E2 proteins, directly interact with PKR and inhibit either its binding to viral dsRNA or its activation. Overexpression of human papillomavirus E6 protein was reported to induce PKR localization to P-bodies where it is sequestered (Hebner et al., 2006). Viruses, such as adenoviruses and Epstein Barr virus produce small RNA inhibitors of PKR (Langland et al., 2006, Sharp et al., 1993). However, a recent report suggests that adenovirus also overcomes PKR activation by an alternative viral protein-mediated mechanism (Spurgeon and Ornelles, 2009). Indirect mechanisms include recruitment of cellular p58ipk by the influenza NS1 protein into a complex with PKR where it binds to the PKR dimerization interface preventing activation (Lee et al., 1990) and recruitment of PP1a by the HSV protein γ134.5 to dephosphorylate eIF2a (He et al., 1998). Consistent with our previous data showing that WNV Eg101 infection does not induce significant eIF2a phosphorylation in BHK cells (Emara and Brinton, 2007), PKR phosphorylation was not significantly induced in rodent cells after infection Caspase-3/7 Inhibitor with either WNV Eg101 or other “natural” lineage 1 or 2 WNV strains. The activation of PKR in cells infected with many other types of viruses resulted in the evolution of viral-mediated processes to suppress PKR activation or activity. Evidence for a WNV-mediated mechanism of PKR suppression was not found. Instead, the results indicate that even though some WNV dsRNAs can activate PKR in vitro, WNV has developed a means to hide its dsRNA from PKR both at early and late times of the infection cycle so that PKR is not activated in infected cells.