Mutagenesis versus Inhibition in the Efficiency of Extinction of Foot-and-Mouth Disease Virus

Abstract

RNA viruses replicate near the error threshold for maintenance of genetic information, and an increase in mutation frequency during replication may drive RNA viruses to extinction in a process termed lethal mutagenesis. This report addresses the efficiency of extinction (versus escape from extinction) of foot-andmouth disease virus (FMDV) by combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H). Selection of G-or H-resistant, extinctionescape mutants occurred with low-fitness virus only in the absence of FU and with high-fitness virus with some mutagen-inhibitor combinations tested. The combination of FU, G, and H prevented selection of extinctionescape mutants in all cases examined, and extinction of high-fitness FMDV could not be achieved by equivalent inhibitory activity exerted by the nonmutagenic agents. The G-resistant phenotype was mapped in nonstructural protein 2C by introducing the relevant mutations in infectious cDNA clones. Decreases in FMDV infectivity were accompanied by modest decreases in the intracellular and extracellular levels of FMDV RNA, maximal intracellular concentrations of FU triphosphate, and a decrease in the intracellular concentrations of UTP. In addition to indicating a key participation of mutagenesis in virus extinction, the results suggest that picornaviruses provide versatile experimental systems to approach the problem of extinction failure associated with inhibitor-escape mutants during treatments based on enhanced mutagenesis.The error rate during template copying by viral RNA-dependent RNA polymerases and reverse transcriptases is close to the maximum tolerable for the maintenance of the genetic information of the virus. This is supported by theoretical predictions (reviewed in references 14 and 15) and experimental results on the adverse effects of enhanced mutagenesis on infectivity during cytolytic or persistent infections of viruses as diverse as picornaviruses (1, 9, 10, 23, 35, 52), retroviruses and retroviral vectors (29,30,37), the rhabdovirus vesicular stomatitis virus (VSV) (23), the flavivirus GB virus B (27), the arenavirus lymphocytic choriomeningitis virus (LCMV) (21, 46), and Hantaan virus (50).Virus extinction by enhanced mutagenesis was accompanied by increases in mutant spectrum complexity, as quantitated by mutation frequency and Shannon entropy (1,21,35,46,52). LCMV showed a very high sensitivity to extinction by 5-fluorouracil (FU) during persistent infections in BHK-21 cells (21, 46), and recently it has been shown that administration of FU to mice prevented the establishment of a persistent LCMV infection (46), providing the first evidence of feasibility of a lethal mutagenesis approach to control viral infections in vivo.Studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have documented that low viral relative fitness and low viral load favored FMDV extinction by enhanced mutagenesis (35,52). The effect of fitness was evidenced using FMDV clo...

Suppression of viral infectivity through lethal defection

“…These genomes include cellular structures and viral proteins (i.e., polymerase and structural proteins of the capsid and envelope). Because of the high error rates of RNA viruses, even inside a single cell, the genetic diversity can be very large, because mutants with a reduced ability to replicate or encode functional proteins are continuously generated (12). However, as long as functional products that can act in trans are accessible for any genome in the cellular interior, there is nothing limiting their use by defective mutants unable to code for correct proteins themselves.…”

“…This phenomenon is known as error catastrophe. The high mutation rate of RNA viruses places viral quasispecies very close to the error threshold (5,6), and it has been experimentally demonstrated that small increases of mutation rate through the use of mutagens can force the extinction of viral populations (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Classical theories on error catastrophe equal fitness to replicative ability and explain extinction on the basis of the accumulation of many deleterious mutations in the viral genomes, so that they eventually fail to replicate themselves.…”

Tempo and mode of inhibitor–mutagen antiviral therapies: A multidisciplinary approach

“…It has been shown that a combination of mutagenic agents and antiviral inhibitors is an efficient way to drive FMDV to extinction (13,14). The mutagen succeeds in raising the fraction of defective and lethal mutants in the population, thus decreasing its overall fitness.…”

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.…”