Antiviral Effect and Virus-Host Interactions in Response to Alpha Interferon, Gamma Interferon, Poly(I)-Poly(C), Tumor Necrosis Factor Alpha, and Ribavirin in Hepatitis C Virus Subgenomic Replicons

Robert Lanford

Bernadette Guerra

Helen Lee

Devron Averett

Department of Virology and Immunology, Southwest National Primate Research Center, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227,^{Anadys Pharmaceuticals, San Diego, California 92121}²

^{Corresponding author. Mailing address: Department of Virology and Immunology, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227. Phone: (210) 258-9445. Fax: (210) 670-3329. E-mail:}gro.rbfs.suraci@drofnalr.

Received 2002 Aug 12; Accepted 2002 Oct 22.

Abstract

The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-α), IFN-γ, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-α, IFN-γ, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-α and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

Abstract

Chronic hepatitis C virus (HCV) infections are one of the leading causes of liver disease worldwide (2). The prevalence of HCV infections is 1 to 2%, although certain geographical regions, age groups, and ethnic groups have much higher rates of infection (3). Although symptoms may be mild for decades, 20% of persistently infected individuals may eventually develop serious liver disease including cirrhosis and liver cancer (2). HCV infection is the leading cause for liver transplantation in the United States (12). Although the initial use of interferon (IFN) for treatment of chronic infections yielded marginal results, the current therapeutic regimen of pegylated alpha 2b IFN (IFN-α2b) and ribavirin provides substantially improved rates of sustained viral clearance of 42 and 82% for genotype 1 and genotype 2 and 3, respectively (45). Treatment of acute infections with standard IFN therapy without ribavirin is highly efficacious and approaches 100% sustained viral clearance (33). Nonetheless, a great need exists for improved antiviral agents, since many patients still do not benefit from IFN therapy, and IFN therapy is associated with undesirable side effects. The lack of a suitable tissue culture system has previously hampered the development of antiviral agents, but the recent development of a replicon system for HCV (43) has partially fulfilled this need.

HCV is a member of the Flaviviridae family. The genome is single-stranded, positive-sense RNA (Fig. (Fig.1).1). Since the cloning of the viral genome (1, 11), rapid advances have been attained in defining viral functions (reviewed in references 5 and 56). The 5′ noncoding region (NCR) contains an internal ribosome entry site (IRES). The amino terminus of the viral polyprotein contains the structural proteins, the capsid and two envelope proteins, E1 and E2. The function of p7 is not known. NS2/NS3 is a metalloprotease that cleaves NS2 from NS3. NS3 is a serine protease and the viral helicase. NS4A is a cofactor for the serine protease. The function of NS4B is unknown. NS5B is the viral RNA polymerase. NS5A is a phosphoprotein that contains a sequence known as the IFN sensitivity-determining region (ISDR). Enomoto et al. (17) first demonstrated a relationship to sequence variation in this region and resistance to IFN therapy. Gale and colleagues have shown that this region interacts with PKR, providing a plausible mechanism for the modulation of the host response to IFN (22, 23). However, the precise function of NS5A is still unknown, and whether PKR binding accounts for viral resistance to IFN is controversial (54, 61). NS5A induces interleukin 8 synthesis that may contribute to IFN resistance (25, 55), and NS5A has been shown to interact with grb2 (62) and a SNARE-like protein (66). E2 has been shown to interact with PKR and may be involved in IFN resistance as well (51, 65). Following the polyprotein open reading frame is a 3′ NCR that has a variable region, a long poly(U)-polypyrimidine stretch, and a highly conserved 98-nucleotide terminus.

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FIG. 1.

Schematic of HCV genome and replicon design. The HCV genome is depicted with the 5′ NCR containing an IRES and the 3′ NCR including a variable region (Var), a polyuridine or polypyrimidine stretch (U/PP), and a 98-nucleotide (nt) conserved region (CR). The depiction of the HCV IRES was adapted from the structural study of Honda et al. (28). The open reading frame of the polyprotein is depicted as a rectangle with demarcation of the individual viral protein domains, and the positions of some of the viral functions are depicted above. c, capsid protein; e1 and e2, envelope proteins E1 and E2. The structure of the bicistronic replicon is illustrated below the HCV genome, with the HCV IRES and EMCV IRES regulating translation of the neomycin phosphotransferase (NEO) gene and the nonstructural proteins of HCV, respectively. A T7 promoter is fused to the HCV 5′ terminus for the production of synthetic RNA. A domain of the NS5A protein is expanded to illustrate the ISDR, PKR binding domain, and the region of the most common adaptive replicon mutations, including the deletion from amino acids 2207 to 2254 observed by Blight et al. (8). The amino acid changes for the adaptive mutations detected in the resident replicons isolated from 10 independent G418-resistant colonies are indicated at the bottom of the figure, and the sites of NS5A hyperphosphorylation (amino acids 2197, 2201, and 2204) are indicated by arrows. WT, wild type.

Although HCV does not replicate in conventional tissue culture systems, a surrogate system has been created on the basis of a bicistronic replicon constructed by Lohmann and coworkers (43). The 5′ end of the HCV genome, including the IRES and 12 codons of the core protein, is fused in frame with the neomycin phosphotransferase gene, the encephalomyocarditis virus (EMCV) IRES drives translation of the HCV nonstructural proteins, and the construct terminates with the 3′ NCR of HCV. When synthetic RNA from this construct is transfected into the human liver cell line Huh7, G418-resistant colonies which persistently maintain the replicon RNA can be isolated. The utility of this system was markedly enhanced when Blight and coworkers (8) demonstrated that adaptive mutations arise that permit highly efficient colony formation. This has been reproduced in a number of studies including this one (8, 27, 35, 42). A replicon has been developed from a second genotype 1b strain (27, 32) that did not require adaptive mutations for high colony-forming efficiency (32), and recently replicons containing the full-length genome have been developed (32, 53).

In this study, we have utilized the replicon system for analysis of several compounds for potential antiviral effect, including IFN-α, IFN-γ, tumor necrosis factor alpha (TNF-α), poly(I)-poly(C), and ribavirin. Although no antiviral effect was observed with TNF-α or poly(I)-poly(C), a synthetic double-stranded RNA (dsRNA) and known inducer of IFN and IFN-stimulated genes (ISGs), both IFN-α and IFN-γ exhibited antiviral effects. The antiviral effect of ribavirin in this system could be ascribed to the induction of error-prone replication similar to recent findings with GB virus B (GBV-B) (38), a surrogate model for HCV. The expression levels for a number of ISGs were monitored before and after antiviral treatments to begin a characterization of the virus-host interactions involved in this system.

Acknowledgments

This work was supported in part by NIH grants U19 AI40035, RO1 AI49574, and P51 RR13986.

We thank Stuart Ray and David Thomas for insightful discussions on the potential antiviral role of ribavirin once the replicating viral population has been reduced by IFN treatment.

Acknowledgments

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