16th International AIDS Conference Toronto, Canada - August 13 - 18, 2006

ATTACHMENT IN HIV ENTRY PROCESS BY ELECTRON MICROSCOPIC TOMOGRAPHY

Int Conf AIDS. 2006 Aug 13-18;16 Abstract No. MoAa0101

Goto T.¹, Hasegawa T.², Kajimura N.², Iwabu Y.³, Yamamoto D.⁴, Ikuta K.³, Takaoka A.^2
1Kyoto University, School of Health Science, Faculty of Medicine, Kyoto, Japan, ²Osaka University, Research Center of Ultra-High Voltage Electron Microscopy, Osaka, Japan, ³Osaka University, Department of Virology, Research Institute for Microbial Diseases, Osaka, Japan, ⁴Osaka Medical College, Biomedical Computation Center, Osaka, Japan

BACKGROUND: After the interaction of viral spikes and receptors on the cell membrane, infection is established. It is important for the prevention and treatment of HIV infection to clarify the whole process of entry. Structural analysis at the nanoscale level is advancing using automated efficient electron microscopy and image analysis. In this study, we tried to clarify the attachment of HIV to the cell membrane in the entry process using electron microscopic tomography.

METHODS: HIV was inoculated into culture lymphocytes, and after a designated time the cells were harvested, fixed and embedded for electron microscopy. The sections were observed with an electron microscope attached to a tilting apparatus. Images were automatically recorded with a CCD camera. The recorded image data were subjected to 3-D construction processing for tomography.

RESULTS: In electron microscopic tomography, there are two competing conditions for obtaining a detailed restructuring image without any distortion: it is necessary to use a wide-range of observation angles with the tilting apparatus and to minimize electron beam damage. To meet these two requirements, we used a slit grid and an automated electron microscope. Although it is very difficult to find the point of contact of the virus and the cell (minimum distance) directly by conventional electron microscopy, with this technique we could easily find the point where the virus contacted the cell membrane. The detailed structure of the contact point is further analyzed and identified at the molecular level using molecular models with three dimensional position data from the Protein Database of HIV envelope and CD4 molecules.

CONCLUSIONS: At the first contact point, the distance between the virus and the cell membrane corresponds very well to the length of the extended gp41 molecule. Further stages have been investigated and will be discussed.

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2006-08-13
MoAa0101

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