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 initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular 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 terminus 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), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. 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 experiment 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 translation of the HCV RNA genome.