Translational insensitivity to potent activation of PKR by HCV IRES RNA

Translation of hepatitis C virus (HCV) is initiated at an internal ribosome entry site (IRES) located at the Tend of its RNA genome. The HCV IRES is highly structured and greater than 50% of its nucleotides form based-paired helices. We report here that the HCV IRES is an activator of PKR. an interferon-induced enzyme that participates in host cell defense against viral infection. Binding of HCV IRES RNA to PKR leads to a greatly increased (20-fold) rate and level (4.5-fold) of PKR autophosphorylation compared to previously studied dsRNA activators. We have mapped the domains in the IRES required for PKR activation to domains III-IV and demonstrate that the N-terminal double-stranded RNA binding domains of PKR bind to the IRES in a similar manner to other RNA activators. Addition of HCV IRES RNA inhibits cap-dependent translation in lysates via phosphorylation of PKR and eIF2 alpha. However, HCV IRES-mediated translation is not inhibited by the phosphorylation of PKR and eIF2 alpha. The results presented here suggest that hydrolysis of GTP by eIF2 is not an essential step in IRES-mediated translation. Thus, HCV can use structured RNAs to its advantage in translation, while avoiding the deleterious effects of PKR activation. (C) 2009 Published by Elsevier B.V.