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.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 1.

Sequence alignments for replicating RNA sequences corresponding to selected HCV genomic regions under untreated and treated conditions. RNAs were harvested, reverse transcribed, cloned, and independently sequenced. (A) Alignment of cDNA sequences of E1/E2 HVR-1 and NS5B (the only variant regions) following T7/full-length H77 transfection under untreated conditions. (B) Alignment of 5′ UTR, core, E1/E2 HVR-1, NS5A, and NS5B cDNA sequences under treatment with RBV at 50 or 400 μM or with IFN-α at 100,000 IU. Alignments were performed and compared with the parent H77 sequence. Nonsynonymous and synonymous nucleotide substitutions are indicated. (C and D) Alignment of 5′ UTR, core, and E1/E2 HVR-1 sequences following T7/ΔBglII transfection under untreated conditions (C) or under treatment with 50 μM RBV (D). Alignments were performed and compared with the parent H77 sequence.

FIG. 2.

(A) RNase protection assay and Western blot analysis of RBV dose effects (in micromolar concentrations) on HCV negative-strand (top panel) and core protein (bottom panel) synthesis under conditions of HCV RNA replication in CV-1 and HepG2 cells. RBV at 400 μM produced moderate inhibition of HCV negative-strand RNA synthesis in both cell types (data not shown). (B) RNase protection assay and Western blot analysis of RBV dose effects (in micromolar concentrations) on T7-dependent β-galactosidase RNA (top panel) and protein (bottom panel) synthesis under conditions of HCV RNA replication in CV-1 and HepG2 cells. pOS8, β-galactosidase expression plasmid.

FIG. 3.

Numbers of mutations from all tested regions of the HCV genome (5′ UTR, core, E1/E2, NS5A, and NS5B) for T7/H77 RNAs that were either left untreated (T7 no tx) or treated either with RBV at 50 or 400 μM or with IFN. Numbers of synonymous (solid) and nonsynonymous (open) nucleotide substitutions are shown for each condition.