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

Abstract

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 inhibiting mRNA translation initiation through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). 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 E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVDeltaE3L) resulted in increased phosphorylation levels of both PKR and eIF2alpha. IFN-pretreated cells infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2alpha in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVDeltaE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2alpha and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVDeltaE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.

FIG. 1

NS5A reduces virus-induced PKR autophosphorylation and eIF2α phosphoryltion. (A) Expression of NS5A in VVNS5A-infected cells. Lysates (20 μg of protein) from HeLa S3 cells pretreated with IFN-α/β and mock-infected (lane A) or infected with wild-type (WT) VV (lane B), VVΔE3L (lane C), or VVNS5A (lanes D through F) at 2, 4, or 6 h postinfection (p.i.) were resolved by SDS-PAGE (14% gel). For mock infections and infections with wild-type VV or VVΔE3L, only samples from the 6-h time point are shown. NS5A was detected by Western blotting (WB) using an anti-NS5A antibody (ID Labs). Arrows indicate the various migrating forms of NS5A. Sizes are indicated in kilodaltons. (B) Transient inhibition of virus-induced PKR autophosphorylation by NS5A. Lysates (20 μg of protein) used for panel A were resolved by SDS-PAGE (7.5% gel). PKR (top panel) and actin (bottom panel) proteins were detected with an anti-PKR antibody and an anti-actin (ICN) antibody, respectively. Hyperphosphorylated PKR protein is denoted by an asterisk. (C) Reduced eIF2α phosphorylation in the presence of NS5A. Lysates were immunoblotted with an antibody specific to the Ser51-phosphorylated form of eIF2α (top panel) or an anti-eIF2α antibody to detect protein levels (bottom panel). The relative levels of eIF2α phosphorylation were determined by quantitative densitometry and normalized against the individual total protein amounts. The ratio of the phospho-specific signal to the total protein signal is indicated below the lanes for lysates from VVΔE3L- and VVNS5A-infected cells. ND, not detectable.

FIG. 2

Inhibition of p38 activation and eIF4E phosphorylation by NS5A expression in VV-infected cells. (A) Virus-induced p38 activation in HeLa S3 cells infected with different recombinant VV at various time points postinfection (p.i.). Lysates (20 μg of protein) were fractionated by SDS-PAGE. The activation state of p38 was then determined by Western blotting (WB) using antibody specific for the dually phosphorylated activated form of p38 (Thr180/Tyr182) (top panel) or the total p38 protein kinase (bottom panel). The position of activated p38 is indicated by an arrow. The relative levels of p38 phosphorylation were determined by quantitative densitometry and normalized against the individual total protein amounts, and the phospho-specific signal/total protein signal ratios are indicated below the lanes for lysates from VVΔE3L- and VVNS5A-infected cells. ND, not detectable; WT, wild type. (B) Inhibition of Ser209 phosphorylation of eIF4E by NS5A expression in VV-infected cells. Phosphorylation levels of eIF4E at Ser209 were measured by Western blotting of the lysates used for Fig. 1 by using a phospho-specific antibody (top panel), while protein levels of eIF4E were determined by using an anti-eIF4E antibody (bottom panel). The relative levels of eIF4E phosphorylation were determined by quantitative densitometry and normalized against the individual total protein amounts. The ratio of the phospho-specific signal to total protein signal is indicated below the lanes.

FIG. 3

Inhibition of EGF-induced phosphorylation of p38 and eIF4E in an inducible stable NS5A-expressing HeLa cell line system. Tet-Off HeLa cells cultured in the presence (−NS5A) or in the absence of tetracycline (+NS5A) were mock- or EGF-treated (20 ng/ml for 2 min), washed, and incubated for 4 h prior to lysis. The activation state of p38 (A) and Ser209 phosphorylation of eIF4E (B) were then determined by immunoblotting using antibody directed against the phosphorylated forms of the proteins as described in the legend to Fig. 2. Total protein levels were determined as described in the legend to Fig. 2. WB, Western blotting.

FIG. 4

NS5A partially reverses the translational arrest phenotype observed in VVΔE3L-infected cells. (A) Profiles of global protein synthesis in recombinant VV-infected cells. HeLa S3 cells were pretreated with IFN-α/β and mock-infected or infected with wild-type (WT) VV, VVΔE3L, or VVNS5A. Cells were pulse-labeled with [³⁵S]Met and harvested at 2, 4, and 6 h postinfection (p.i.) Lysates (20 μg of protein) were resolved by SDS-PAGE and detected by autoradiography. The protein translation levels were also quantified by subjecting lysates to TCA precipitation followed by scintillation counting. (B) Profiles of viral protein synthesis in recombinant VV-infected cells. The lysates used for panel A were immunoprecipitated with a rabbit polyclonal antiserum against total VV proteins, and precipitated proteins were subjected to SDS-PAGE and autoradiography.

FIG. 5

NS5A-mediated reversal of the translation arrest phenotype observed in VVΔE3L-infected cells is independent of its ability to bind Grb2. Analyses of global protein synthesis in HeLa S3 cells pretreated with IFN-α/β and infected with a recombinant VV expressing a Grb2 binding-defective form of NS5A (VVPro3) were performed as described in the legend to Fig. 4. WT, wild type; p.i., postinfection.