Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System

Abstract

High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid "error catastrophe." Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5 untranslated region (5 UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3 cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5 UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.Genetic variation provides a selective advantage for RNA virus populations, promoting escape from immune selection and rapid adaptation to novel environments (8). The absence of proofreading-repair mechanisms in RNA replicases and transcriptases is thought to contribute to mutation rates in the range of 10 Ϫ3 to 10 Ϫ5 substitutions per nucleotide per round of RNA replication (32). However, two factors exert a counterbalancing pressure against excessive genetic variation. A high degree of conservation of viral genomic sequences must be maintained for interactions with specific cellular proteins, as in the case of internal ribosomal entry. Excessive error rates can also contribute to net loss of fitness by leading to "error catastrophes" that threaten the viability of populations of quasispecies present in viral swarms. While the RNA genome of hepatitis C virus (HCV) contains hypervariable regions that are thought to contribute to immune escape, little is known about their intrinsic origins or their control in the absence of immune or drug selection.The extraordinary genetic diversity of HCV is reflected in its in vivo generation of quasispecies, whic...

Mechanisms of action of interferon and ribavirin in chronic hepatitis C: Summary of a workshop

“…In studies using HCV replication models, mutagenic effects of ribavirin have been identified by several 54,[56][57][58][59][60] but not all authors. 61 When infectivity was used as a functional readout for mutagenesis, diminished infectivity was found with ribavirin treatment of HCV cultures.58 , 60 The concentrations of ribavirin needed to achieve mutagenic effects were high (up to 400 uM) and may exceed those achieved in vivo.…”

Analysis of Ribavirin Mutagenicity in Human Hepatitis C Virus Infection

“…A similar effect of high concentrations of ribavirin has been found in cell cultures infected by RNA viruses belonging to several families, including foot-and-mouth disease virus, Hantaan virus, West Nile virus, and GB virus B, a member of the Flaviviridae family that is closely related to HCV (1,9,16,20,44). In addition, ribavirin has been reported to exert an antiviral effect related to errorprone replication in HCV subgenomic replicons, both in Huh7 cell lines and in a full-length binary HCV replication system (5,20,21,48,53). However, error-prone replication is not the mechanism by which ribavirin acts against arenavirus lymphocytic choriomeningitidis virus or the yellow fever virus, another member of the Flaviviridae family that is related to HCV (24,42).…”

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

“…Incorporated ribavirin causes transition mutations (A-to-G, G-to-A) because of its ability to pair with both uracil and cytosine. Ribavirin has been shown to increase error frequency for both poliovirus genomic RNA and hepatitis C replicon synthesis (1)(2)(3). Thus, ribavirin is thought to exert its antiviral activity by increasing the error rate of viral genome replication past the point of ''error catastrophe,'' whereby the fitness of all members of the viral population is reduced.…”