A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.
Author summary The pig is considered to be a “mixing vessel” for influenza viruses because it can be infected by both human and avian influenza viruses. This mixing of viruses could potentially produce novel recombinant strains that are more adaptable to humans or other mammals. The permissive mechanism that allows pig cells to be infected with avian influenza virus is largely unknown. Here we reveal that the pig cellular protein ANP32A has a unique 106V/156S signature, different from that of ANP32A proteins from other mammals, enabling the protein to bind strongly to avian influenza polymerase at the post-entry step, and promoting avian virus replication. This species-specific 106V/156S epistasis of swANP32A likely determines the susceptibility of pigs to avian influenza infection. Our findings provide novel insights into the molecular basis of interspecies transmission of avian IAV between chickens and pigs.