A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Abstract

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

“…In this study, the several FMDV high-fidelity variants that we isolated with ribavirin exhibited restricted broad-spectrum mutagen resistance compared with the R84H variant. All these results suggest that restricted resistance to mutagens for high-fidelity variants is the exception, while broad resistance is the standard, as has been demonstrated by the ribavirin-isolated FMDV M296I variant showing restricted resistance (49). It was believed that the ability of the isolated RdRp variants to identify natural or unnatural nucleotides was associated with the resulting structural changes of the mutant polymerases.…”

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

“…In addition, a number of mutant polymerases that show altered polymerization fidelity have been identified harboring mutations remote from the active site (26)(27)(28). All of these observations suggest that nucleotide binding and incorporation are modulated by a long-distance network of interactions (14,(29)(30)(31)(32).…”

Response of Hepatitis C Virus to Long-Term Passage in the Presence of Alpha Interferon: Multiple Mutations and a Common Phenotype

“…Virus titers were determined by calculating the TCID 50 /ml (48). Briefly, cell culture supernatants were serially diluted and used to infect Huh-7.5 cells that had been seeded in 96-well plates at 6,400 cells/well 16 h earlier.…”

“…The toxicity of IFN-␣ was measured in Huh-7.5 reporter cells by seeding 96-well plates at 70% confluence and exposing the cells to up to 10 8 IU/ml IFN-␣ for 72 h. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well at a final concentration of 500 g/ml; the cells were incubated 4 h, 100 l of dimethyl sulfoxide (DMSO) was added, and the optical density was measured at a wavelength of 550 nm. The CC 50 (the concentration of IFN-␣ that results in cytotoxicity for 50% of the cells) was calculated from four different determinations as described previously (49), and it was higher than 10 8 IU/ml for Huh-7.5 reporter cells. The 50% inhibitory concentration (IC 50 ) of IFN-␣ with respect to HCV infectivity was determined by seeding Huh-7.5 reporter cells in 24-well plates at 70% confluence and exposing the cells to serial dilutions of IFN-␣ (0.1 to 100 IU/ml) for 72 h. IC 50 s were measured using the virus titration as described above and calculated relative to the untreated control levels (defined as 100%).…”