| Hepatitis C virus (HCV) requires numerous host cofactors for its replication, some of which interact with conserved RNA structures. Most of the identified RNA structures are located within the 5' and 3' untranslated regions (UTR). Although it is clear that these RNA structures are required for viral replication, the functions of many of them in viral replication are still unclear. Following up a previous study where we identified eight host cofactors that bind HCV RNA as being required for HCV replication, we sought to further define the role of these cofactors in the HCV life cycle. We developed two luciferase based reporter assays for investigating HCV IRES-mediated translation and RNA replication. Using these assays in conjunction with siRNA silencing, we show that host microRNA miR-122 and polypyrimidine tract-binding protein (PTB) are required for HCV IRES-mediated translation. RPL22, DDX3X, ELAVL1, and HNRNPC are required for HCV replication, but not translation. Surprisingly, silencing EIF2S3, which was reported to be required for HCV translation in vitro, does not impact HCV translation in cells but inhibits its replication. This suggests a translation-independent function of EIF2S3 in HCV replication.;We next further examined the role of the EIF2 complex in HCV replication. We find that silencing EIF2S3 and EIF2S2, but not EIFS1, inhibits HCV replication, but not HCV translation. Using an siRNA trans-complementation assay, we observe that both wild type EIFS2 and a mutant EIFS2 defective in its GTPase activity that is required for translation can complement the defect in HCV replication. Thus, EIF2S3 has a translation-independent function in HCV replication. Interestingly, EIF2S3 and EIF2S2 are required for HCV replication in the genomic context, but are dispensable for replication of bicistronic HCV replicons. The addition of an encephalomyocarditis virus (EMCV) IRES into the HCV genome removes the dependence on EIF2S3 for HCV replication. This suggests that bicistronic HCV replicons, which have been used in the majority of HCV replication studies, do not faithful reproduce all aspects of HCV replication.;One prominent HCV RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stem loop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem loop structure in the 3' UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production, but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and co-localize with sites of HCV replication. EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE and this is dependent on the 5BSL3.2 loop. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication. |
