Enhanced genome replication activity of pandemic H1N1 influenza A virus through PA mutations

The 2009 pandemic H1N1 (pH1N1) influenza A virus (IAV) is a reassortant virus with two polymerase components, PA and PB2, originating from avian IAV. Avian IAV polymerase does not function efficiently in mammalian cells without host-adaptive mutations. The mechanism by which pH1N1 replicates in human hosts is not fully elucidated, as pH1N1 does not contain the host-adaptive PB2 E627K mutation required for species-specific interaction with ANP32, which facilitates replicase (polymerase oligomer) formation. Our previous research revealed that mutations in PA played a key role in mammalian host adaptation of pH1N1. These mutations were found in two separate domains of PA, the C-terminal (CTD) and N-terminal domains (NTD). We reported that the NTD mutations increase the expression of NP through enhanced association of GRSF1 with the mRNA transcripts. However, the role of CTD mutations, which are located at the interface of the polymerase oligomers, has not been elucidated. In this study, we characterized the effect of key CTD mutations and found that the CTD mutations enhanced genome replication activity and replicase formation in vitro. Unexpectedly, rescued viruses containing only the CTD mutations that enhance genome replication activity had an attenuated viral growth phenotype. However, the introduction of an additional NTD mutation to the virus restored virus growth in mammalian cells. These results suggest that the mutations found in the PA NTD are required together with CTD mutations for balanced genome replication and growth in human cells.IMPORTANCEThe 2009 pandemic H1N1 (pH1N1) influenza A virus (IAV) is a reassortant virus with two polymerase components, PA and PB2, originating from avian IAV, which typically does not function well in mammalian cells. All the human IAVs, except pH1N1, contain E627K in the PB2 subunit, which allows the virus to utilize host factor ANP32 to form polymerase oligomers required for genome replication. The mechanism of how pH1N1 adapted to humans and caused seasonal epidemics is not yet fully elucidated, but our previous studies revealed that mutations in PA play a key role in host adaptation. Here, we describe a novel mechanism of host adaptation where mutations in the PA CTD enhance genome replication, whereas PA NTD mutations increase nucleoprotein production to support increased replication activity. This finding highlights the range of mechanisms of host adaptation, which is vital to understanding when assessing the potential emergence of novel viruses.