inosine monophosphate dehydrogenase ͉ IFN regulatory factor

Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.Hepatitis C virus (HCV) is a significant cause of morbidity and mortality, infecting over 170 million people worldwide (1,8). Despite recent progress, the current method of treatment for HCV infection remains inadequate for the majority of patients (24,29,39). HCV is an enveloped, positive-sense, single-stranded RNA virus (25). Its 9.6-kb genome encodes a single polyprotein, which is proteolytically processed by a combination of host-and virus-encoded proteinases into 10 structural and nonstructural (NS) proteins (16,26).The establishment of the HCV subgenomic replicons and the subsequent studies of them revealed that most of the HCV NS proteins are involved in HCV RNA replication (2,15,28,35). NS3 is a helicase and a serine protease whose function is dependent on NS4A. Chimpanzee studies showed that the enzymatic activities of NS3-4A serine protease and NS3 helicase are essential for the productive replication of HCV (21). Thus, these enzymatic activities may be key components in the replication of HCV subgenomic replicons. NS4B is an integral membrane protein which induces intracellular membrane alterations (9, 18). NS4B may play an important role in the formation of the HCV RNA replication complex (14a). NS5A is also involved in HCV RNA replication, as adaptive mutations in NS5A are associated with increased replication efficiency of HCV RNA (2, 22, 27). Its membrane association through its N-terminal transmembrane domain is essential for RNA replication of the subgenomic replicon (11). NS5B is an RNA-dependent RNA polymerase, a key enzyme involved in viral replication (21). NS5B contains a hydrophobic domain (21 amino acids [amino acids 571 to 591]) at its C terminus, which is a tr...