Influence of Salt Bridges in the Avian Influenza Virus HA on Acid-Induced Membrane Fusion and Pathogenicity

The influenza virus hemagglutinin (HA) is known to play an essential role in virus infection. It is involved in cell receptor binding and uptake by endocytosis and, after internalization and trafficking to early and late endosomes, induces membrane fusion leading to the release of the viral genome into the cell. This fusion process is mediated by a water-driven “spring- loaded mechanism” which is initiated by protonation of the HA1 globular heads in the acidic environment of the endosome. It has been shown for the HA of influenza virus X-31 that the pH of fusion can be shifted to higher or lower pH values by introducing or deleting salt bridges in the ectodomain of the glycoprotein. In particular, a mutation at the distal end of HA1 leading to the introduction of a salt bridge between two monomers significantly enhanced its stability thereby abolishing membrane fusion. It was suggested that this mutation is also involved in the development of new human pathogenic H5N1 lineages. We hypothesize that salt bridges in the HA1 ectodomain regulate replication efficiency and pathogenesis. As a proof of principle recombinant virus particles containing the wild type or mutant HA of X-31 strain were produced and assayed by fluorescence dequenching. Fusion activity of mutant HA was significantly reduced with increasing pH compared to the wild type, suggesting an influence of the salt bridge on viral fusion. We will apply these results on avian HA and measure virus-endosome fusion by real time imaging inside living cells. Using human and avian cells we will be able to observe possible variations in endosomal pH and in membrane fusion activity. This work was supported by the Marie Curie Actions 7th framework program of Initial Training Networks.