In Vitro RNA Replication Directed by Replicase Complexes Isolated from the Subgenomic Replicon Cells of Hepatitis C Virus

Abstract

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg 2؉ and Mn 2؉ ) showed different effects on RNA synthesis. Mg 2؉ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn 2؉ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3 deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3 dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.Hepatitis C virus (HCV) is the major cause of non-A, non-B transfusion-associated hepatitis and accounts for a significant proportion of viral hepatitis cases worldwide (9, 18). Although HCV infection is resolved in some subjects, up to 80% of infected individuals develop chronic HCV infection. HCV is a positive-sense RNA virus belonging to the Flaviviridae family (5). The life cycle of HCV consists of several important processes that occur primarily in the cytoplasm of the host cells. Among these important processes, RNA replication is a key step in the viral proliferation cycle and is thus an important target for antiviral development. The genome of HCV consists of approximately 9,600 bases, with a single open reading frame encoding a polyprotein of around 3,010 amino acids. The open reading frame is flanked by 5Ј and 3Ј untranslated regions, which are important for translation and RNA replication. The polyprotein is cleaved both co-and posttranslationally by cellular and virally encoded proteases into at least 10 different structural and nonstructural proteins (1, 9, 18).The NS5B protein of HCV has been shown to possess RNAdependent RNA polymerase activity in vitro. However, recombinant NS5B alone lacks the strict template specificity required for RNA synthesis (2, 6-8, 11, 13-15, 21, 27, 28). The lack of template specificity is presumably due to the absence of other viral or host factors that constitute the viral replicase complexes (RCs) in vivo. It is believed that RCs...

Redox regulation of hepatitis C in nonalcoholic and alcoholic liver

“…The results are consistent with previous reports that, too, have demonstrated suppression of HCV replication by other divalent cations such as zinc [198][199][200][201]. In addition, Stat-3 participates in the anti-HCV activity of interferon [197,202,203], and the HCV-induced oxidative stress has recently been suggested to inhibit HCV RNA replication instead, by activating the gene expression of COX-2 and increasing the production of prostaglandin E2 [73].…”

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

“…To distinguish these possibilities, we designed experiments to separate viral transcription from viral translation. We performed an in vitro replication assay using HCV RCs in crude membrane fractions of the replicon cells [31,33,43,44]. Furthermore, we performed immunodepletion of PTB from the lysates to assess the effects of PTB in RNA replication in vitro.…”

“…These results together support the notion that the active HCV RCs existed in DRM structure that consists of lipid rafts. On the other hand, Lai et al [44] showed that the RNA replication activity of HCV RC was inhibited by a lower concentration of NP-40. The difference in the detergent sensitivity of HCV RNA replication between our studies and the other report may be attributed to the proportion of NS proteins existing in DRM fraction.…”

“…They could simply prevent PTB from being incorporated into the HCV RCs, or they could interfere with its function after assembly of the HCV RCs. Lai et al [44] demonstrated that HCV RC preparation could not use exogenous RNA as template and that the newly synthesized single-strand RNA could serve as the template for an additional round of RNA synthesis. HCV proteins have been shown to be relatively stable [60].…”

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

“…Interestingly, Lai et al recently showed that the HCV RNA replicase complex isolated from the subgenomic replicon cells can synthesize both double-stranded RNA products and single-stranded RNA product served as a template for further round of RNA synthesis. This indicates the possibility that HCV replicase can also undergo cyclic replication in vitro when the other nonstructural viral proteins are present in the HCV RNA replicase complex (Lai et al, 2003). Since the cellular factors present in the replicase complex have not been identified, in vitro reconstitution for HCV RNA replicase still requires analyses of functional cellular and/or viral proteins directly involved in the replication process.…”