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J. Biol. Chem., Vol. 281, Issue 18, 12729-12735, May 5, 2006

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Membrane Fusion by Single Influenza Hemagglutinin Trimers

KINETIC EVIDENCE FROM IMAGE ANALYSIS OF HEMAGGLUTININ-RECONSTITUTED VESICLES^*

Masaki Imai, Takafumi Mizuno, and Kazunori Kawasaki¹

From the Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 and the Department of Virology III, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan

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, HA₀, at various HA_1,2:(HA_1,2 + HA₀) 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 HA₀) 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-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.

Received for publication, January 30, 2006

^* This work was funded by National Institute of Advanced Industrial Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4 and Tables S1 and S2.

¹ To whom correspondence should be addressed: Tel.: 81-29-861-6602; Fax: 81-29-861-6147; E-mail: kawasaki.k{at}aist.go.jp.

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