Abstract
Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis. The surface density of hemagglutinin fusion-activity sites on the vesicles was varied, while keeping the surface density of receptor-binding activity sites constant, by co-reconstitution of the fusogenic form of hemagglutinin, HA(1,2), and the non-fusogenic form, HA(0), at various HA(1,2):(HA(1,2) + HA(0)) ratios. The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy. The best fit of a log-log plot of fusion rate versus the surface density of HA(1,2) exhibited a slope of 0.85, strongly supporting the hypothesis that single hemagglutinin trimers are sufficient for fusion. When only HA(1,2) (without HA(0)) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA(1,2) was distinct from that for the HA(1,2)-HA(0) co-reconstitution. The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers.
Highlights
Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general
To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis
The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy
Summary
The receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. When only HA1,2 (without HA0) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA1,2 was distinct from that for the HA1,2-HA0 co-reconstitution The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers. One series of experiments involved co-reconstitution of fusogenic HA1,2 trimers and non-fusogenic HA0 trimers with various HA1,2:(HA1,2 ϩ HA0) ratios and with a constant protein/lipid ratio to assess the effect of the surface density of the fusion-activity sites of HA without changes in the surface density of the receptor-binding sites. The other series of experiments involved reconstitution of HA1,2 trimers without HA0 trimers, in which the surface densities of the fusion-activity sites and the receptor-binding sites were changed in parallel. The former series of experiments allowed us to deduce the minimum number of HA trimers required for fusion, whereas the latter series, in comparison with the former, could be used to evaluate how much HA-receptor binding interfered with fusion
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