Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Abstract

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. IMPORTANCEInfluenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. I nfluenza virus is a single-stranded, negative-sense RNA virus with a genome consisting of 8 segments (1). Like other RNA viruses, influenza virus replicates with extremely low fidelity. Its RNA-dependent RNA polymerase (RdRp) complex, which includes the viral proteins PB1, PB2, PA, and NP (2, 3), has a mutation rate of approximately 2.3 ϫ 10 Ϫ5 substitutions per nucleotide per cell ...

Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis

“…Second, the presence of a natural ribose implies that in theory, chain elongation is possible in the case that T-705-RTP or RBV-TP is used as an alternative substrate by the influenza virus polymerase. These two factors could explain recent observations that cell culture passaging of influenza virus in the presence of T-705 (25) or ribavirin (26,27) leads to virus mutagenesis; this mutagenic effect was also seen in influenza virus-infected mice receiving T-705 therapy (28). Enzymatic studies showed that influenza virus polymerase recognizes T-705-RTP as an alternative substrate versus GTP and ATP (29,30).…”

A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro

“…Substitutions that confer resistance to polymerase inhibitors would be expected to significantly decrease virus fitness (74,75). Serial passage in cells with various nucleoside analogues, such as ribavirin, T-705, 5-azacytidine, and 5-fluorou-racil, often fails to yield specific substitutions in the polymerase proteins because of the lethal mutagenesis mechanism of these compounds (76)(77)(78). However, this mechanism is different from that employed by RO-7; it is unknown to what degree the viral PA protein can escape from RO-7 drug pressure and still retain suitable endonuclease activity.…”

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

“…In short, we used overlap PCR mutagenesis to introduce the following mutations: HA, T1583G; HA, G1006T; HA, G542T; M, T861G; M, A541C; NA, G1168T; NA, C454T; NP, A454C; NP, A1160T; NS, G227T; NS, A809G; PA, T964G; PA, T237A; PA, A1358T; PB1, G599A; PB1, G1764T; PB1, T1288A; PB2, A1854G; PB2, A440T; and PB2, A1167T. Viruses were rescued from transfected cells as described previously (38).…”

“…Madin-Darby canine kidney cells were provided by Arnold S. Monto (University of Michigan School of Public Health) and were maintained in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) with 10% fetal bovine serum (Gibco and HyClone), 25 mM HEPES (Invitrogen), and 0.1875% bovine serum albumin (Life Technologies). Influenza A/WSN/33(H1N1) virus was rescued from transfected cells using an 8-plasmid reverse genetic system containing each genomic segment (pHW181 to -188), a kind gift from Robert Webster (St. Jude's Children's Research Hospital) (37,38). A biological clone of influenza A/Puerto Rico/8/1934(H1N1) virus was obtained from the ATCC (VR-1469), and the genomic segments were cloned into the pHW2000 reversegenetic system (38).…”

“…Influenza A/WSN/33(H1N1) virus was rescued from transfected cells using an 8-plasmid reverse genetic system containing each genomic segment (pHW181 to -188), a kind gift from Robert Webster (St. Jude's Children's Research Hospital) (37,38). A biological clone of influenza A/Puerto Rico/8/1934(H1N1) virus was obtained from the ATCC (VR-1469), and the genomic segments were cloned into the pHW2000 reversegenetic system (38). The sequences of these clones were verified using Sanger sequencing.…”