Impaired host shutoff is a fitness cost associated with baloxavir marboxil resistance mutations in influenza A virus PA/PA-X nuclease domain

The polymerase acidic (PA) protein is a subunit of the trimeric influenza A virus (IAV) RNA-dependent RNA polymerase and the target of the anti-influenza drug baloxavir marboxil (BXM). As with other direct-acting antivirals, treatment with BXM can lead to selection of viruses carrying resistance mutations. If these mutations have negligible fitness costs, resistant viruses can spread widely and render existing treatments obsolete. Multiple BXM resistance mutations in the nuclease domain of PA have been identified, with I38T and I38M amino acid substitutions occurring frequently. These mutations have minimal to no effects on viral polymerase activity, virus replication, or transmission. However, for reasons that are not well understood, viruses with BXM resistance substitutions have not been able to compete with parental wild-type strains. The IAV genome segment encoding PA also encodes the host shutoff nuclease PA-X, which shares the endonuclease domain with PA but has a unique C-terminal domain generated by ribosomal frameshifting during translation. Unlike their effects on PA activity, the effects of BXM or the I38T/M substitutions on PA-X function remain uncharacterized. In our work, for the first time, we directly examine the effects of baloxavir and the I38T/M substitutions on PA-X activity and show that baloxavir inhibits PA-X activity in a dose dependent manner. Most importantly, we also demonstrate that the I38T/M mutations significantly impair the host shutoff activity of PA-X proteins from different IAV strains of H1N1, H3N2, and H5N1 subtypes. Our work reveals that the deleterious effects of I38T/M on PA-X function may represent an important barrier to the spread of BXM-resistant viruses.