Molecular determinants of HCV replication

Studies of pestivirus replication have been facilitated over the past five years by the development of replicon systems, as pioneered by others and us. This led to new insights into the roles of viral and host elements in virus replication. For example, using the replicon of bovine viral diarrhea virus (BVDV), we identified the viral nonstructural proteins that are necessary and sufficient to catalyze viral RNA replication and established whether the proteins operate in cis or trans during the assembly of the functional replication complex. Moreover, we defined RNA motifs in the untranslated regions of the viral genome that are essential for translation, replication or both processes, respectively, and found that binding of the cellular NFAR group of proteins to these motifs plays a key role in RNA replication. These data have lead to a model of the initial steps of the pestivirus RNA replication pathway that involves a circular conformation of the BVDV genome (52). Extension of the replicon approach to the BVDV related hepatitis C virus (HCV) was recently achieved (17, 83). As a result, it is now possible to study HCV RNA replication in detail, building initially on similarities and differences to the much better characterized BVDV replicon. Thus, expanding our studies to HCV, we recently found that the NFAR proteins also bind to HCV RNA, and RNAi and co-localization studies suggested that members of this group of proteins represent essential components of the HCV replication complex. In this proposal we consider complementary experimental approaches with HCV and BVDV, to gain further insights into the molecular details that determine the viral replication process.