The C-Terminal Transmembrane Domain of Hepatitis C Virus (HCV) RNA Polymerase Is Essential for HCV Replication In Vivo

Abstract

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...

Kissing-Loop Interaction in the 3′ End of the Hepatitis C Virus Genome Essential for RNA Replication

“…Care was taken for the codon usage, because the introduction of rare codons might interfere with proper RNA translation, resulting in premature translation termination and C-terminally truncated NS5B proteins. Since the C-terminal membrane anchor of NS5B that is encoded in this region is essential for RNA replication, the presence of rare codons might interfere with our studies (24,32). Finally, the mutations were tested for their impact on disturbing the SL structures by using the Mfold algorithm (data not shown).…”

Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication

“…This demonstrates that the replication defect of this mutant was due to a loss of protein function and not to an effect on overlapping RNA functions. While this paper was in preparation, a report by Lee et al (19) described replication defects of NS5B mutants with a deletion of the insertion sequence or an introduction of two stop codons at aa 571. However, it was not experimentally validated whether these mutations affected the function of the overlapping CRE.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…1). 67,68 Similar to many other polymerases, 69 NS5B is composed of finger, thumb and palm domains, but has a tightly encircled active site and an additional allosteric GTP binding site. 70 The finger and thumb subdomains form a tunnel through which the single-strand RNA is guided to the active site, while the NTPs enter the active site via a second positively charged tunnel.…”