NA enzymatic activity was eliminated despite in Vero cells cotransfected with pCAGGS HA and NA plasmids by treatment with oseltamivir carboxylate (3, 51) (Fig. (Fig.4B).4B). The syncytium formation assay was repeated using cells coexpressing the HA and NA proteins in the presence of oseltamivir carboxylate. When NA enzymatic activity was inhibited by the drug, the pH of HA-mediated membrane fusion decreased to pH 5.5, the same value observed in cells expressing HA protein alone (Fig. (Fig.4A).4A). These results are consistent with the promotion of H5 HA-mediated membrane fusion by N1 neuraminidase activity. FIG. 4. Contribution of NA enzymatic activity to the pH of membrane fusion mediated by the HA protein. (A) Representative photomicrographs of syncytia showing the contribution of the NA protein to HA-mediated membrane fusion.

The pH values are given in the top … DISCUSSION To investigate how the pH of activation of the HA protein influences the in vitro and in vivo properties of influenza viruses, we compared four recombinant viruses with altered pH-dependent HA protein stability to wild-type A/chicken/Vietnam/C58/04 (H5N1) virus. An N1142K mutation in the HA2 fusion peptide pocket region increased the activation pH of the HA protein from 5.9 to 6.4, allowing activation under mildly acidic conditions. This mutation dramatically reduced the fitness of the virus in three ways: (i) multiple-step replication in vitro was reduced by a factor greater than 10; (ii) infectivity in the environment decreased four times as rapidly as that of wild-type virus; and (iii) the virus reverted to the wild-type sequence within 5 passages in chicken eggs and after inoculation in mallards.

The N1142K mutation may increase the pH of activation of the HA protein above the threshold pH at which a significant portion of intracellularly cleaved HA trimers become prematurely triggered, and inactivated, during transport to the cell surface (44). The HA protein mutations Y231H and K582I changed the activation pH of the HA protein to 6.3 and 5.4, respectively. While these two mutations had opposite effects on the activation pH, the recombinant viruses bearing the mutations had similar phenotypes. Despite in vitro replication rates similar to that of wild-type virus, the viruses bearing a Y231H or K582I mutation did not induce weight loss, neurological signs, or mortality in mallards, were not efficiently transmitted, and were shed significantly less.

Overall, the data show that efficient and sustainable infection of mallards by H5N1 influenza virus is not supported by HA protein activation pH values less than 5.5 or greater than 6.2. The results Brefeldin_A of experiments with the virus bearing an HA-H241Q mutation suggest that robust infection in mallards is supported by activation pH values between 5.6 and 5.9.