Virus-Host Cell Interactions during Hepatitis C Virus RNA Replication: Impact of Polyprotein Expression on the Cellular Transcriptome and Cell Cycle Association with Viral RNA Synthesis

Abstract

Hepatitis C is the most common cause of clinically significant liver disease in the United States. Although acute infection with hepatitis C virus (HCV) is usually subclinical, persistent infection develops in up to 80% of initially infected patients (2). The majority of persistently infected individuals develop evidence of chronic liver injury and are at risk for progressive hepatic fibrosis leading to cirrhosis and ultimately death due to liver failure or hepatocellular carcinoma. Approximately 10,000 individuals die each year with chronic liver disease associated with HCV infection in the United States alone (1), rendering this virus a very significant public health problem.HCV is classified within a distinct genus, Hepacivirus, in the family Flaviviridae (15). Its genome consists of positive sense, single-stranded RNA of approximately 9.5 kb in length (9). The genomic RNA contains a single open reading frame, from which one large polyprotein is expressed, and flanking 5Ј and 3Ј untranslated regions. Translation of the viral RNA occurs through a cap-independent mechanism via an internal ribosomal entry site (IRES) located in the 5Ј untranslated region. A combination of cellular and virally encoded protease activities leads to processing of the polyprotein into individual structural and nonstructural proteins. Three structural proteins have been identified with the core protein forming a poorly defined viral capsid, and E1 and E2, both glycosylated envelope proteins, forming heterodimers. These structural proteins are processed by host signal peptidases. C terminal to E2, further proteolytic processing by host peptidases yields a short hydrophobic peptide, p7, which may function as a membrane channel (44), followed by the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS2 and the amino-terminal protease domain of NS3 are responsible for proteolytic cleavage at the NS2-NS3 junction (20, 52), whereas other proteolytic cleavage events involved in the maturation of the NS proteins are carried out by the major viral serine protease, NS3, in association with its cofactor, NS4A (5,7,35). In addition to its function as a protease, NS3 also contains helicase and nucleoside triphosphatase activities within its C-terminal domain (28). Specific replicative functions have not been identified for NS4B and NS5A, but both are likely to contribute to the viral replicase complex. NS5B constitutes the HCV RNA-dependent RNA polymerase and is thus the catalytic core of the replicase.In addition to functions related to RNA replication, both NS3/4A and NS5A appear to play important roles in modulating antiviral responses within infected cells and in promoting long-term persistence of the virus. The NS3/4A protease blocks

Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase

“…We have shown that HCV RNA synthesis is stimulated during the S phase of the cell cycle (13). Moreover, compared with capdependent translation, the cap-independent translation of HCV RNA is enhanced in actively growing cells and reduced in resting cells (29).…”

“…We studied two independent, Huh7 hepatoma cell lines, NNeo͞ C-5B clones 2-3 and 3, both of which contain replicating genome-length RNA expressing all of the proteins of the HCV-N strain (12). We compared the abundance of Rb in these cell lines with that in their progeny, designated 2-3c and 3c, respectively, in which HCV RNA had been eliminated by prior treatment with IFN-␣2b (13). The abundance of Rb in each of the HCV RNA-containing cell lines was reduced compared with that in the cognate-cured cell line (Fig.…”

“…Prior studies have failed to show any significant difference in the regulation of cell-cycle progression in the 2-3 replicon cells and their cured 2-3c counterparts (13). However, the Huh7 cells from which these cells were derived are known to contain a p53 mutation and express little p21 (24,25).…”

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

“…We compared the abundance of ectopically expressed TRIF-Flag in Huh7 2-3 cells, which express NS3͞4A from a replicating genome-length RNA, and in clonally related 2-3c cells from which the viral RNA had been eliminated by prior IFN-␣2b treatment (29). Quantitative immunoblot analysis indicated that TRIF-Flag abundance was reduced Ϸ50% in the 2-3 cells compared with the cured 2-3c cells (Fig.…”

Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

“…Huh7-HP, Huh7-A7, and Huh7-K2040 are Huh7 cells that harbor culture-adapted variants of the Con1 HCV (genotype 1b) subgenomic replicon RNA and have been described in detail (3,6,9). Huh7 2-3 and Huh7 2-3c are Huh7-derived cell lines that harbor the full-length self-replicating HCV genome (derived from the HCV-N strain, genotype 1b) and their IFN-cured counterparts, respectively (3,10,11). UNS3͞4A (clone 24) are osteosarcoma cells that conditionally express an HCV 1a NS3͞4A construct under control of a tetracycline-regulated (tet-off) promoter (a gift from D. Moradpour, University of Lausanne, Lausanne, Switzerland).…”