Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation i nitiation in a cap-independent manner. Previously, we reported the interact ion between La antigen and the HCV IRES, which appeared to occur in the con text of initiator AUG, It was further shown that HCV IRES-mediated translat ion was stimulated in the presence of human La antigen. In this study, we h ave defined the cis- and transacting elements responsible for La-5'-NCR int eractions and established the dependence of the HCV IRES efficiency on cell ular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C t erminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), gen erated against La protein that selectively binds La in HeLa lysates and com petes for the protein binding to the 5'-NCR, was used to demonstrate the re quirement of La for the HCV IRES function in the context of mono- and dicis tronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translat ion lysates blocked the HCV and poliovirus IRES-mediated translation in vit ro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experim ent in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of tran slation of the HCV RNA genome.