Watching Influenza get Hogtied

Influenza viruses have hundreds of hemagglutinin (HA) protein trimers embedded in their phospholipid envelope. The HA protein is the lynchpin in the first stage of influenza infection, first by facilitating binding and internalization into target host cells and then by mediating fusion with the membrane of late endosomes. Recently, antibodies capable of neutralizing both group 1 and group 2 influenza A viruses have been described [Science,v324,p246,2009; Science,v333,p843,2011]. These broadly neutralizing HA stem-binding antibodies appear to act by blocking the HA conformational change necessary for fusion. Here, we have visualized the interplay between a virus and antibodies during neutralization using our recently developed single-particle fusion assay [PNAS,v105(40),p15382,2008]. To this end, antibodies and infectious viral particles were fluorescently labeled and incubated together. Viruses were then bound to sialic acid decorated proteins incorporated into a planar, supported phospholipid bilayer and fusion induced by addition of an acidic buffer. Using TIRF microscopy, individual hemifusion events were observed as the rapid release of lipophilic dye from the viral membrane into the target bilayer. This novel approach allows us to directly link the action of individual HA-binding antibodies to viral fusion events, providing new insight into the mechanism and molecular stoichiometry involved in fusion and neutralization - information obfuscated in traditional cell-cell and bulk fusion assays. We see a concentration dependent effect of inhibiting antibodies on the time it takes for viruses to undergo hemifusion as well as on the total number of hemifusion events. Furthermore, we can correlate the fluorescence intensity per virion to the number of bound antibodies and thereby directly measure the number of HA molecules involved in fusion. More to the point, our results have explicit implications for the model of bilayer fusion arising from the coordinated efforts of multiple hemagglutinins.