The mechanism of resistance to favipiravir in influenza

Abstract

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

Keywords: antiviral; influenza; polymerase; resistance; virus.

Fig. 1.

Experimental evolution leads to resistance to favipiravir following 10 passages. (A) Virus inhibition assay of Eng195 by favipiravir. Supernatants were titered following 48 h of growth on MDCK cells. Error bars are ±SD, n = 3. (B) Virus inhibition assay with 3.5 µM favipiravir for Eng195 and passage 10 populations. Error bars are ±SD, n = 6, one-way ANOVA with Dunnett’s multiple comparison test, ***P < 0.001.

Fig. 2.

K229R and P653L combine to give resistance to favipiravir in a minigenome assay. (A) Minigenome assay with Eng195, PB1 K229R, PA P653L, and PB1 K229R + PA P653L in presence of increasing concentrations of favipiravir. Polymerase activity is given as Firefly/Renilla. (B) Relative polymerase activity to no drug of data presented in A. Error bars are ±SD, n = 3, two-way ANOVA with Tukey’s honestly significant difference (HSD), ***P < 0.001.

Fig. 3.

PB1 K229R has a cost to viral growth that is rescued by PA P653L. (A) Viral growth curves on MDCKs at MOI = 0.002 for Eng195, PB1 K229R, PA P653L, and PB1 K229R + PA P653L. (B) Virus inhibition assay for Eng195 and PB1 K229R + PA P653L in presence of increasing concentrations of favipiravir. Error bars are ±SD, n = 3, one-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001.

Fig. 4.

PB1 K229R confers resistance to favipiravir in other influenza A polymerases. (A) Minigenome assay with H3N2 A/Victoria/3/1975, PB1 K229R, PA P653L, and PB1 K229R + PA P653L in presence of increasing concentrations of favipiravir. (B) Minigenome assay with H7N9 A/Anhui/1/2013, PB1 K229R, PA P653L, and PB1 K229R + PA P653L in presence of increasing concentrations of favipiravir. Error bars are ±SD, n = 3, two-way ANOVA with Tukey’s HSD, ***P < 0.001.

Fig. 5.

PB1 K229R does not confer resistance to ribavirin but prevents mutations caused by favipiravir. (A) Minigenome assay showing relative polymerase activity for Eng195 and K229R + P653L in presence of increasing concentrations of ribavirin. Error bars are ±SD, n = 3, two-way ANOVA. (B) Individual mutations per 10,000 sequenced nucleotides above the control (0 µM, Eng195) measured by primer ID at 0, 10, and 100 µM favipiravir from RNA expressed in a minigenome assay with Eng195, PB1 K229R, PA P653L, or PB1 K229R + PA P653L polymerase. n = 2, two-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, ***P < 0.001.

Fig. 6.

Resistance to favipiravir triphosphate incorporation by the influenza A virus RNA polymerase in vitro. (A) Schematic showing how a radiolabeled capped primer (red) binds the 3′ terminus of the 3′ “4C template.” Initiation can start at 3′ G3 of the template giving the G product or at 3′ C2 giving the C product. (B) Total polymerase activity of the wild-type influenza A virus polymerase and the three mutants on the 3′ 4C template quantified by combining C and G products. (C and D) Gel analysis and quantitation of the activity of the wild type or PB1 K229R mutant influenza A virus RNA polymerase on the 3′ 4C template provided with CTP and either GTP or 500 μM F-RTP. The radiolabeled capped primer signal is indicated with P. (E) Schematic showing how an ApG dinucleotide primer (gray) binds the 3′ terminus of “promoter up” template. Initiation starts at 3′ A3 of the template by the incorporation of UTP (+1 product). The first [α -³²P]GTP is incorporated opposite C11 (+9 product). (F) Gel analysis of the replication activity of the wild type or PB1 K229R mutant polymerase on the promoter up template in the absence or presence of 500 μM F-RTP. (G) Quantitation of the replication assay performed with wild type, K229R, P653L, or double mutant polymerase. Error bars are ±SD, n = 3, one-way ANOVA with Dunnett’s multiple comparison test in B and G, and two-sided t test in D, *P < 0.5, ***P < 0.001, ns, not significant.

Fig. 7.

Alignment and structural modeling of the PB1 K229R mutation. (A) Sequence alignments of PB1 and PA subunits of the influenza virus RdRP. Mutations present in favipiravir-resistant virus indicated by black arrows. (B) Cartoon model of F-RTP binding the influenza A virus RdRp. Motifs C and F are indicated as well as the helix of the PA C terminus (PA-C) that contains residue P653. (C) Surface models of F-RTP binding by the wild-type influenza A virus and by the K229R mutant. The location of motif C is indicated.