Regulation of mRNA translation and cellular signaling by hepatitis C virusnonstructural protein NS5A

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR), PKR mediates the host IFN-induced antiviral response at least in part by inhibi ting mRNA translation initiation through phosphorylation of the or subunit of eukaryotic initiation factor 2 (eIF2 alpha). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3 L protein is a potent inhibitor of PKR, Accordingly, infection of IFN-pretr eated HeLa S3 cells with an E3L-deficient VV (VV Delta E3L) resulted in inc reased phosphorylation levels of both PKR and eIF2 alpha, IFN-pretreated ce lls infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2 alpha in a t ransient manner. We also observed an increase in activation of p38 mitogen- activated protein kinase in IFN-pretreated cells infected with VV Delta E3L , consistent with reports that p38 lies downstream of the PKR pathway. Furt hermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Impo rtantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal gro wth factor-treated cells stably expressing NS5A, NS5A-induced inhibition of eIF2a and eIF4E phosphorylation may exert counteracting effects on mRNA tr anslation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a pa rtial and transient restoration of cellular and viral mRNA translation comp ared with IFN-pretreated cells infected with VV Delta E3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a pot ential mechanism by which HCV might maintain global mRNA translation rate d uring early virus infection while favoring cap-independent translation of H CV mRNA during late infection.