Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.

Abstract

Hepatitis C virus (HCV) is the major etiological agent of non‐A, non‐B post‐transfusion hepatitis. Its genome, a (+)‐stranded RNA molecule of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)‐strand RNA viruses, the replication of HCV is thought to proceed via the initial synthesis of a complementary (‐) RNA strand, which serves, in turn, as a template for the production of progeny (+)‐strand RNA molecules. An RNA‐dependent RNA polymerase has been postulated to be involved in both of these steps. Using the heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA‐dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non‐structural protein 5B (NS5B). The characterization of the HCV RNA‐dependent RNA polymerase product revealed that dimer‐sized hairpin‐like RNA molecules are generated in vitro, indicating that NS5B‐mediated RNA polymerization proceeds by priming on the template via a ‘copy‐back’ mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA‐ or DNA oligonucleotide primer‐dependent RNA synthesis on templates with a blocked 3′ end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

“…RNA product size increased steadily up to 120 min when the product reached the template size ( Figure 3A, lane 6). The kinetics of RNA synthesis using a full-length HCV RNA template over 360 min timecourse indicated that RNA synthesis is not initiated from the 3'-snap-back structure, in contrast to previous reports using TNTase-associated HCV NS5B proteins purified from insect cells (Behrens et al, 1996;Lohmann et al, 1997;Ishii et al, 1999). Next, we investigated whether HCV NS5B can perform cyclic replication without help of cellular and/or viral factors; synthesis of complementary RNA using the input RNA template and subsequent use of the RNA products for the synthesis of original input RNA template.…”

“…In addition, insect cell-expressed full-length NS5B generated approximately template-size labeled X-RNA by action of TNTase associated with NS5B (Ishii et al, 1999). Other previous studies have also shown that HCV NS5B proteins expressed in insect cells are associated with host TNTase (Behrens et al, 1996;Lohmann et al, 1997). The NS5B with TNTase activity generated dimer-size RNA product using the full-length 9.6-kb HCV RNA template, which is probably the RNA product synthesized by TNTase-mediated priming and/or copy-back RNA synthesis mechanism from the X-RNA.…”

“…Previously, insect cell-expressed full-length NS5B proteins have been shown to initiate RNA synthesis by snap-back intramolecular priming (Behrens et al, 1996;Lohmann et al, 1997) or by internal initiation in the poly(U) and 3'-UTR of the HCV genome (Sun et al, 2000;Pellerin et al, 2002), whereas the full-length HCV RNA polymerase expressed in E. coli was shown to start RNA synthesis de novo from internal single strand region in X-RNA loop I and from the 3'-end of (-) 3'-UTR of HCV genome (Blight and Rice, 1997;Oh et al, 1999). To clarify this discrepancy, RdRp reactions was performed using X-RNA and the (-) 3'-UTR of HCV.…”

Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines

“…NS5B has been identified as an RNA-dependent RNA polymerase that is responsible for the synthesis of negative-strand HCV RNA. 48 Inhibition of NS5B has been long considered an attractive target for therapeutic intervention in HCV-infected patients. 48,49 Our data suggest that NS5B may have a role in interfering IFN signaling pathway.…”

“…48 Inhibition of NS5B has been long considered an attractive target for therapeutic intervention in HCV-infected patients. 48,49 Our data suggest that NS5B may have a role in interfering IFN signaling pathway. Future studies are needed to examine the mechanism involved in NS5B-mediated inhibition of the IFN signaling pathway.…”