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© The Rockefeller University Press, 0021-9525/1997//1041 $5.00
The Journal of Cell Biology, Volume 137, Number 5, , 1997 1041-1056

Article

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy



Toku Kanaseki*, Kazunori Kawasaki{ddagger}, Masayuki Murata§, Yoko Ikeuchi*, and Shun-ichi Ohnishi§

* Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183; {ddagger} Cellular Biophysics Laboratory, National Institute of Bioscience and Human Technology, Tsukuba 305; and § Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or "microprotrusions of the lipid bilayer." At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the "initial fusion pore" at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.

Abbreviations used in this paper: ESR, elecron spin resonance; HA, hemagglutinin; IMP, intramembrane particle; NA, neuroaminidase.

Address all correspondence to Toku Kanaseki, The Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu City, Tokyo 183, Japan. Tel.: 81-423-25-3881. Fax: 81-423-21-8678.

Shun-ichi Ohnishi's present address is The Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University Seta Campus, 1-5 Yokoya, Seta Ohe-Cho, Ohtsu, Shiga 520-21 Japan.


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