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.

Importance: Influenza 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.

FIG 1

Sensitivity of influenza virus to nucleoside analogs. MDCK cells were infected with influenza A/PR8/34 (●), A/WSN/33 (◼), A/Panama/2007/1999 (◆), or A/Wyoming/03/2003 (▼) virus at an MOI of 0.1 in the presence of nucleoside analogs at the indicated concentrations (x axis). Cells were treated with ribavirin (A), 5-azacytidine (B), or 5-fluorouracil (C). Titers in supernatants were determined at 24 h and are shown relative to the 0 μM drug control. Solid lines, H1N1 strains; dashed lines, H3N2 strains. Points are plotted as mean ± standard deviation for 3 replicates.

FIG 2

Effect of nucleoside analogs on MDCK cells. (A) Number of viable cells relative to mock-treated controls after 24 h of drug treatment at the indicated concentrations (x axis) as analyzed by MTT assay. Each point represents the mean ± standard deviation for 3 replicates. (B) Cytotoxicity was measured using the CytoTox-Glo protease release assay on cells plated at low density and treated for 24 h with nucleoside analogs. Percent cytotoxicity (y axis) is expressed relative to untreated cells. (C) Images of cells after treatment with the indicated nucleoside analogs for 24 h. The drug concentrations shown are the highest used in any of the experiments involving influenza virus that are described in the text. Magnification, ×20.

FIG 3

Mutation frequency in influenza virus populations treated with nucleoside analogs. MDCK cells were infected with influenza A/PR8/34 (H1N1) virus at an MOI of 0.1 in drug-containing medium. Supernatants were harvested at 24 h postinfection. A 957-base fragment of the HA gene was amplified and cloned. Between 51 and 110 clones from each sample were sequenced, and mutations were identified. Overall mutation frequencies are expressed per 10⁴ bases sequenced. Wild-type bases are on the left in each table. Specific mutation types are expressed per 10⁴ wild-type bases sequenced. Mutations identified in multiple clones were counted once. A chi-square test was used to determine the statistical significance of the differences in total mutation frequency for each mutation type relative to the no-drug control. *, P < 0.05; **, P < 0.005. Statistically significant increases are highlighted by shading. (A) Each nucleoside analog compared to a no-drug control. (B) Treatment with multiple concentrations of ribavirin.

FIG 4

Specific infectivity of influenza virus populations treated with nucleoside analogs. MDCK cells were infected with influenza A/PR8/34 (H1N1) virus at an MOI of 0.1 and treated with ribavirin (A), 5-azacytidine (B), or 5-fluorouracil (C). Supernatants were harvested at 24 h postinfection, and titers of infectious virus were determined by TCID₅₀ assay. Quantitative reverse transcription-PCR was used to determine the genome copy number in the samples. This was used to calculate the specific infectivity (TCID₅₀/genome copy), which is shown relative to the 0 μM drug sample. Statistical significance was determined using the Kruskal-Wallis test with a Dunn correction. *, P < 0.05; **, P < 0.005. Points are plotted as mean ± standard deviation for 4 replicates.

FIG 5

Effect of IMPDH inhibition on influenza virus. MDCK cells were treated with ribavirin or mycophenolic acid either with or without 40 μM guanosine and infected with influenza A/PR8/34 (H1N1) virus at an MOI of 0.1. At 24 h postinfection, culture supernatants were harvested and used for both determination of infectious virus titers by TCID₅₀ assay and quantitative reverse transcription-PCR. Infectious titer (A) and specific infectivity (TCID₅₀/genome copy) (B) data are shown normalized to 0 μM drug. Specific infectivities were compared to the 0 μM drug samples using the Kruskal-Wallis test with a Dunn correction. *, P < 0.05. Solid lines, samples treated with drug only; dashed lines, samples with drug plus 40 μM guanosine. Points are plotted as mean ± standard deviation for 4 replicates.

FIG 6

Lethal mutagenesis of influenza virus. Influenza A/PR8/34 (H1N1) virus was passaged on MDCK cells in ribavirin (A), 5-azacytidine (B), or 5-fluorouracil (C). Cells were infected at each passage with an MOI of ≤0.1 as described in Materials and Methods, and progeny were harvested at 24 h postinfection. Three viral lineages were passaged for each condition. Solid lines, 3 mock-treated control lineages; dashed lines, 3 drug-treated lineages. The horizontal dotted lines indicate the limit of detection for the last passage of each experiment. When titers dropped below the limit of detection, 0.8 ml of supernatant was added to fresh MDCK cells in the absence of drug, and titers were determined at 4 days postinfection. Daggers indicate that no virus was recovered from any of the three lineages at that passage.

FIG 7

Serial passage of influenza virus in sublethal concentrations of nucleoside analogs. Influenza A/PR8/34 (H1N1) virus was passaged on MDCK cells in the presence of nucleoside analogs. Passages were performed at an MOI of 0.01 using supernatant from the previous passage, and cells were harvested at 24 h postinfection. (A) Infectious titers for 16 passages at the indicated drug concentrations. (B) Infectious titers of passage 16 drug-treated and mock-treated (D2) populations after a single passage at an MOI of 0.1 (black bars) or 0.01 (gray bars) over a 24-h period in the absence of nucleoside analogs. (C) Sensitivity of passage 16 populations to the drugs in which they had been passaged. Infectious titers of viruses after a single passage at an MOI of 0.01 in drug for 24 h are shown. Titers are relative to those from virus passaged in the absence of drug. Solid lines with solid symbols, unpassaged and DMSO-passaged controls. Dashed lines with open symbols, viruses that had been passaged in drug. The horizontal dotted line indicates the limit of detection. Points are plotted as mean ± standard deviation for 3 replicates.

FIG 8

Mutation accumulation within viral populations after serial passage in nucleoside analogs. Influenza A/PR8/34 (H1N1) virus was serially passaged in 7.5 μM ribavirin (R1 to R3), 7.5 μM 5-azacytidine (A1 to A3), or 30 μM 5-fluorouracil (F1 to F3), or without drug (D1 to D3). At passage 16, viral populations were sequenced to a high depth of coverage using the Illumina platform. The location, frequency, and type of all mutations above 1% frequency and with a P value of below 0.01 are shown. The influenza virus genome segments are concatenated with positions 1 to 2341 representing PB2, 2342 to 4682 representing PB1, 4683 to 6915 representing PA, 6916 to 8693 representing HA, 8694 to 10258 representing NP, 10259 to 11671 representing NA, 11672 to 12698 representing M, and 12699 to 13588 representing NS. Mutations above the dashed line (frequency of 0.5) are consensus mutations within the population. Red dots, C-to-U and G-to-A transition mutations; blue dots, C-to-G and G-to-C transversion mutations; black dots, A-to-G and U-to-C transition mutations; white dots, all other mutation types, including deletions.