Efficient Virus Extinction by Combinations of a Mutagen and Antiviral Inhibitors

Abstract

An increase in the mutation rate during replication of RNA viruses can result in a decrease of viral infectivity and occasional virus extinction (11,34,39,40,60). Studies with the important animal pathogen foot-and-mouth disease virus (FMDV)-a member of the Picornaviridae family (53, 63)-have shown that a small replicative population size and low viral fitness favored virus extinction (60). This was documented with single and multiple passages of FMDVs differing up to 10 6 -fold in relative fitness in the absence or presence of the mutagenic base analogs 5-fluorourocil (FU) or 5-azacytidine, individually or in combination (59, 60). Mutagenic treatments resulted in occasional, not systematic, viral extinction, while parallel passages in the absence of mutagens never led to loss of infectivity, no matter how low the initial viral population size and fitness were (59, 60). These results suggested the possibility that a combination of an antiviral inhibitor, to reduce the replicative load of virus, and a mutagenic agent could be more effective in producing viral extinction than a mutagenic agent used in isolation. To test this possibility we have studied the effect of the mutagenic base analog FU and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of FMDV clones and populations depicting a wide range of relative fitness values. FU has been shown to be mutagenic for a number of RNA viruses (6,20,31,34,51,71), including FMDV (59,60). G at millimolar concentrations blocks the replication of picornaviruses (5,7,15,49,52,55), arboviruses (27), and several plant viruses (13, 67). In poliovirus, the target of G is the ATPase activity of nonstructural protein 2C (49), a protein involved in viral replication and encapsidation. In FMDV, amino acid substitutions at 2C have also been associated with resistance to G (56). Heparins are sulfated polysaccharides (9) which bind FMDV when the virus has been passaged in cell culture and has adapted to using heparan sulfate (HS) as a receptor (2,35,54). Adaptation to use of HS as a receptor has been associated with substitutions which lead to positively charged amino acids at exposed positions of the capsid (2, 54).Here we report that high-frequency extinctions of FMDV of low and intermediate fitness values can be achieved with a combination of FU and G but not with either drug alone. Extinction of high-fitness FMDV populations required a triple combination of G and H together with FU. Mutation frequencies in the mutant spectrum of three genomic regions of a preextinction population obtained by the combined action of an inhibitor and a mutagen were compared to values in genomes passaged in standard conditions and also to values previously determined for FMDV populations subjected to one or multiple passages in the presence of FU (60). We found that mutation frequencies increased up to fourfold. There was also a statistically significant increase in the number of mutations in preextinction populations with respect to control populations, and the ...

An interfering activity against lymphocytic choriomeningitis virus replication associated with enhanced mutagenesis

“…The types and location of the mutations indicate that ADAR 1-L or related activities were not involved in the production of mutated LCMV genomes (Polson & Bass, 1994;Zahn et al, 2007). The mutational bias found, an abundance of AAG and UAC transitions, coincides with that observed previously in FMDV and LCMV populations treated with FU (Grande-Pérez et al, 2002, 2005bPariente et al, 2001;Sierra et al, 2000). Although the interfering activity was not dependent on the infectivity present in the interfering populations, it was dependent on the dose and age of the supernatant.…”

“…LCMV was considered extinguished when no RT-PCR-amplifiable material and no infectivity (limit of detection ¡33 p.f.u. ml 21 ) could be observed in the cell-culture supernatant after three blind passages of the undiluted viral population in BHK-21 cells in standard culture medium, in the absence of mutagens (Pariente et al, 2001;Sierra et al, 2000). In addition, extinction was also ascertained in a cellular extract of the BHK-21 cells after the first blind passage.…”

Lethal Mutagenesis in Viruses and Bacteria

“…Considerable experimental evidence suggests that high mutation rates in RNA virus populations have powered their rapid evolution (Eggers and Tamm 1965;Domingo et al 1978;de la Torre et al 1990;Domingo 2000). However, artificially elevated mutation rates were shown to have deleterious effects on the fitness of RNA viruses and to eventually lead to extinction of the viral population beyond certain mutation rate thresholds (Loeb et al 1999;Sierra et al 2000;Pariente et al 2001;Grande-Perez et al 2002;Anderson et al 2004;Freistadt et al 2004;Bull et al 2007;Graci et al 2007Graci et al , 2008Zeldovich et al 2007). This observation is called lethal mutagenesis for RNA viruses.…”

Sequential structures provide insights into the fidelity of RNA replication

“…This unique biochemical activity and the high mutation rate of RNA viruses have stimulated the interest in the use of mutagenic rNTP analogs to induce virus entry into error catastrophe. Among them, ribavirin (1-␤-D-ribofuranosyl-1,2,3-triazole-3-carboxamide) and 5-f luorouracil have been shown to drive different RNA viruses to extinction through enhanced mutagenesis, including foot-and-mouth disease virus (FMDV) (4)(5)(6)(7)(8)(9)(10)(11). Structural studies on replicative complexes of viral RDRPs, template-primers, and rNTP substrates or analogs are needed to understand the molecular basis of the low fidelity of copy of these enzymes and the mutagenic activities displayed by rNTP analogs on viral replication.…”

“…The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases. 5-fluorouracil ͉ foot-and-mouth disease virus ͉ replication fidelity ͉ ribavirin ͉ RNA elongation R NA virus populations exist as collections of similar but genetically distinct genomes named quasi-species. Genetic heterogeneity favors adaptation of viruses to environmental changes.…”