Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission

Abstract

Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC ₅₀. EC ₅₀s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA–BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.

Author summary

Baloxavir is a new and potent anti-influenza drug targeting essential functions of viral replication. Currently, the I38T polymerase acidic protein (PA) substitution is the major marker of reduced susceptibility and potential resistance to baloxavir, but the full baloxavir resistance profile remains unclear. Here, we demonstrated that PA E23G/K substitutions alone weaken baloxavir efficacy, but they also synergize with I38T to impair drug activity further. E23G/K substitutions are located close to the binding site of baloxavir and indirectly weaken key drug-binding interactions. This effect has some negative consequences for virus replication, but E23G/K viruses possess the capacity for airborne spread between naïve ferrets, the gold-standard model of human influenza transmission. Therefore, E23G/K viruses have the potential for community spread, which would adversely affect baloxavir clinical implementation. Our study supports ongoing surveillance for circulating human E23G/K viruses, and it may inform design of enhanced baloxavir-like drugs less susceptible to emergence of viral resistance.