H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion

Abstract

The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH 4 Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T49 2 S, N72 2 D, R75 2 K, and S113 2 F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCEContinuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

“…To characterize membrane fusion properties of the recombinant IAVs, we determined the pHs at which the viral HAs underwent an acid-induced conformational transition (pH 50-HA ) and at which the virus lost infectivity owing to acid-induced inactivation of the HA (pH 50-inact ). In addition, we compared levels of inhibition of viral infection by the lysosomotropic agent ammonium chloride, which counteracts the acidification of endosomes and lysosomes; a higher sensitivity to NH 4 Cl reflected a viral requirement for a lower pH during fusion and/or uncoating (24). Collectively, these three assays indicated that the HAs of Mem-H1N1 and the two duck IAVs, mal-H1N1 and mal-H2N2, underwent conformational transition and mediated membrane fusion at a lower pH than the HAs of VN-H5N1 and Sh-H7N9 (Table 1).…”

“…The pH optimum of HA-mediated fusion of avian and mammalian IAVs varies depending on the host species and the history of virus evolution, and our study, for the first time, demonstrates that this variation influences viral sensitivity to IFN and IFITMs. Among different IAVs, human viruses typically have the lowest fusion pH optimum (from 5.0 to 5.4), whereas swine viruses and viruses of gallinaceous poultry, including high-and low-pathogenic IAVs of the H5 and H7 subtypes, display relatively high fusion pH optima (5.6 to 6.0) (22,24,(34)(35)(36)(37)(38)(39). IAVs isolated from wild aquatic birds appear to be particularly variable with respect to the fusion pH optimum (from 5.0 to 6.0), although available data are limited (22-24, 37, 38).…”

“…Inhibition of viral cell entry by ammonium chloride. The assay used to measure inhibition of viral cell entry by ammonium chloride was described in detail elsewhere (24). In brief, MDCK cells in 96-well plates were infected with the viruses in the presence of various concentrations of NH 4 Cl, incubated overnight, fixed, and immunostained for viral NP.…”

“…Inactivation of viral infectivity at acidic pH. The assay used to determine the inactivation of viral infectivity at acidic pH was described elsewhere (24). In brief, viral stocks were diluted in morpholineethanesulfonic acid (MES)-buffered saline at pHs ranging from 5.0 to 7.0, incubated for 15 min at 37°C, neutralized, and titrated with a single-cycle assay in MDCK cells.…”

“…Differences in the receptor binding specificity of animal and human viruses and differential expression of sialic acid receptors in the human respiratory tract determine, to a significant extent, differences in viral cell and tissue tropism, transmissibility, and disease severity in humans (reviewed in references 4 and 19 to 21). Furthermore, there is growing evidence that the interval of acidic pH at which HA undergoes a conformational transition and mediates membrane fusion (pH optimum of fusion) also differs between IAVs from different host species (22)(23)(24), and these differences in the pH optimum of fusion can affect viral interspecies transmission and pathogenicity (reviewed in references 25 to 27).…”