Repression of the PKR protein kinase by the hepatitis C virus NS5A protein: A potential mechanism of interferon resistance

Background: Chronic infection with hepatitis C virus (HCV) is associated with progressive liver damage, including the development of cirrhosis and hepatocellular carcinoma, and HCV is a leading cause of liver dysfunction worldwide. The current therapy for chronic HCV infection, interferon-α (IFN), is effective in a minority of HCV-infected patients. Several studies have demonstrated a correlation between therapeutic outcome and the amino :acid sequence of a small region of the HCV non-structural 5A (NS5A) gene product. It has been suggested that this region, termed the interferon sensitivity-determining region (ISDR), may mediate IFN resistance by directly interacting with one or more cellular proteins associated with the IFN-mediated antiviral response. Objectives: In an attempt to define the molecular mechanism by which the NS5A protein and the ISDR might contribute to HCV resistance to IFN, we examined whether NS5A could regulate the IFN-induced protein kinase, PKR, a primary mediator of the IFN-induced antiviral response. Study design: Multiple approaches, including in vitro assays using recombinant proteins, the transfection of recombinant clones into cultured cells, and in vivo studies in yeast, were used to examine the interaction of NS5A with PKR, as well as the functional significance of the interaction. An ISDR deletion mutant was prepared to evaluate tile importance of the ISDR in mediating the NS5A-PKR interaction and the requirement of this region for PE;R inhibition. Results: NS5A repressed PKR activity through a direct interaction with the protein kinase catalytic domain. Both PE;R repression and interaction required the presence of the ISDR. Conclusions: Inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Thus, therapeutic strategies designed to block the NS5A-PKR interaction may increase the efficacy of IFN therapy in HCV-infected individuals.