A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence

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Abstract
The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N–infected mice compared with wild-type 1918 virus–infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus. PB1-F2 is the most recently discovered protein produced by the influenza A virus. It has been previously shown that PB1-F2 is present in the mitochondria, where it induces cell death; our laboratory has demonstrated that PB1-F2 is a contributor to pathogenesis in the mouse model of infection. To study PB1-F2 further, we examined highly pathogenic strains of avian influenza virus and located an amino acid change that seemed to be associated with increased death in mice. We studied this amino acid change in PB1-F2 at position 66 in two different viruses. A recombinant virus that has a PB1 gene from an H5N1 virus was used as well as a fully reconstructed 1918 pandemic virus. In this study, we show that a mutation in PB1-F2 found in highly pathogenic influenza A virus isolates causes nonpathogenic viruses to induce disease in mice. In addition, we show that the increased pathogenicity is associated with higher levels of virus and cytokines in the lungs. We conclude that PB1-F2 does affect pathogenicity, and that position 66 seems to play an important role in contributing to the effects of PB1-F2 in the mouse model.