The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

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The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-␣(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.