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

Abstract

Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

Fig. 1.

Passage of poliovirus in the presence of ribavirin to generate resistance. (A) Viral passage in 400 μM ribavirin. HeLa cells were infected at an MOI of 0.1 PFU per cell in the presence or absence of 400 μM ribavirin. Resulting titers were graphed as a function of passage number. (B) Viral passage in 100 μM ribavirin followed by 400 μM ribavirin. A total of 100 μM ribavirin was used for passages 1–4, and 400 μM ribavirin was used for passages 5–9. (C) Generation of ribavirin resistance during passaging. The percentage resistance for each of nine passages of pool 3 virus stocks from B was determined by titering in the presence or absence of 800 μM ribavirin. The percentage resistance of a virus isolate, 3-1, obtained by three plaque-purification steps in the presence of 400 μM ribavirin, is shown. The percentage resistance of virus containing the single amino acid change G64S is also shown.

Fig. 3.

Ribavirin-resistance profiles of the selected virus. (A) Plaque assay of G64S and wild-type virus on HeLa cells in the presence of various concentrations of ribavirin. (B) Serial passage of G64S and wild-type virus in the presence or absence of 400 μM ribavirin. HeLa cells were infected at an MOI of 10 PFU per cell in the presence or absence of 400 μM ribavirin. Resulting amounts of virus (PFU per cell) were graphed as a function of passage number. (C) Serial passage of G64S and wild-type virus in the presence or absence of 800 μM ribavirin (performed as in B, in the presence or absence of 800 μM ribavirin).

Fig. 4.

Comparative fidelity of wild-type (WT) and G64S mutant virus. (A) The frequency of wild-type and G64S viruses that also contained mutations conferring resistance to 0.5 mM guanidine. Standard errors from two replicate experiments of the stocks used in Fig. 3 are shown. (B) The frequency of viruses that contained mutations conferring resistance to 0.5 mM guanidine for seven independently derived stocks of wild-type and G64S mutant virus. Seven individual measurements, and the average (avg) with standard error, are shown. (C) Serial passage of wild-type and G64S mutant virus in the presence and absence of 10 μM 5-AZC. Passages in the presence and absence of drug were performed as in Fig. 3.

Fig. 2.

Sequence of the G64S region in separate pools of virus that display ribavirin resistance. Virion RNA was isolated, amplified by RT-PCR, and sequenced from wild-type virus (A), G64S virus (B), a mixture of wild-type and G64S viruses (C), pool 1 passage 9 (D), pool 2 passage 9 (E), and pool 3 passage 9 (F). The sequencing chromatograms are shown. The wild-type sequence is GTG GGT AAC, and the G64S sequence is GTG AGT AAC. Arrows indicate position 6176, the location of the G-to-A transition mutation in G64S.

Fig. 5.

Poliovirus polymerase structure. The known structure of poliovirus polymerase (23) is shown. Catalytic aspartate residues in the active site, 328, 329, and 233 are shown in red, residues thought to contact nucleotide substrates directly are shown in royal blue (31), and Ile-67, the closest resolved residue to Gly-64, is shown in yellow.