Role of Influenza Polymerase in Host Adaptation

Influenza A viruses infect a wide range of hosts, including humans and many avian species. One of the mechanisms for the instigation of a pandemic is direct infection of humans with an avian virus that contains mutations allowing it to infect and spread among humans. Before 1997, direct infection of humans with avian influenza viruses was not considered a threat to human health. However, several cases of human infection with highly pathogenic avian H5N1 influenza viruses were reported in 1997 in Hong Kong showing that direct avian-to-human transmission of influenza can occur. Characterization of the isolated H5N1 viruses indicates that mutations in polymerase gene play a major role in human host adaptation. We did extensive analysis on polymerase proteins to identify mutations that enhance the activity in mammalian hosts. We revealed that PB2 residue 271, which is highly conserved among human viruses contribute for enhanced activity in mammalian cells. This study indicates that there are multiple residues in PB2 that can affect the mammalian host adaptation of avian influenza viruses.

In 2009, a new pandemic influenza virus emerged. The source of the 2009 pandemic virus was a Mexican swine-origin reassortant virus of the H1N1 subtype (pH1N1). Several reassortment steps led to the emergence of this virus, which possesses HA, NP, and NS genes of the classical swine lineage, NA and M from the Eurasian swine lineage, a human PB1 that was seeded from an avian virus in approximately 1968, and avian PA and PB2 genes. The avian-origin PB2 contains 591R, which is thought to compensate for the lack of 627K, and 271A, which we have revealed to enhance polymerase activity in the mammalian hosts. In addition to PB2 mutations, we found that the pH1N1 PA gene is a major factor for high pH1N1 polymerase activity in the mammalian cells. Analysis using site-directed mutagenesis identified several pH1N1 PA residues that enhance avian polymerase activity in the mammalian cells. However, this enhancement was observed at 37°C but not at the lower temperature of 34°C. An additional introduction of pH1N1 PB2 enhanced activity at 34°C, suggesting the presence of unidentified residues in PB2 that are required for efficient growth at the low temperature. Using a reporter gene assay, we identified novel mutations, PB2 V661A and V683T/A684S, which are involved in enhanced pH1N1 polymerase activity at low temperature. The growth of a recombinant pH1N1 containing PB2 with avian virus residues 271T/661V/683V/684A was strongly reduced in human cells compared to wild-type virus at low temperature. Among the four residues, 271A and 684S are conserved in human and pH1N1 viruses but not in avian viruses, suggesting an important role in mammalian adaptation of pH1N1 virus. Our results indicate that these mutations in both PA and PB2 are required for efficient virus replication in mammalian hosts.

Lutz Iv MM; Dunagan MM; Kurebayashi Y; Takimoto T. Key Role of the Influenza A Virus PA Gene Segment in the Emergence of Pandemic Viruses. Viruses 12(4). 2020. PMCID: PMC7232137

Bussey KA, Bousse TL, Desmet EA, Kim B, Takimoto T. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J. Virol. 84:4395-4406, 2010. PMCID: PMC2863787.

Bussey KA, Desmet EA, Mattiacio JL, Hamilton A, Bradel-Tretheway B, Bussey HE, Kim B, Dewhurst S, Takimoto T. PA residues in the 2009 H1N1 pandemic influenza virus enhance avian virus polymerase activity in mammalian cells. J Virol 85:7020-7028, 2011. PMCID: PMC3126589.

Hayashi T, Wills S, Bussey KA, Takimoto T. Identification of influenza A virus PB2 residues involved in enhanced polymerase activity and virus growth in mammalian cells at low temperature. J Virol 89:8042-9, 2015. PMCID: PMC4505657.

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