IVEM TOMOGRAPHY OF HIGH PRESSURE FROZEN & FREEZE SUBSTITUTED VIRUS STRUCTURES

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(Supported by Swedish Medical Research Foundation MFR K9706X1217501; Swedish Foundation for Strategic Research; Wallenberg Research Foundation. P.I. R.H. Cheng) Cyro-EM, in conjunction with X-ray crystallographic data and antibody labeling, has been very effective in addressing biological structure-function issues. The IVEM has the potential for decreased radiation damage, higher resolution, and also serves as a comparative base for contrast-transfer function deconvolution. Several types of large viruses will be imaged, both tilted and un-tilted, by cryo-EM. In addition, plastic-section tomography will be carried out to study membrane interactions during virus budding. During this reporting period, the focus was on the budding process and the structure of the virus core particle in the cytoplasm and during budding. BHK cells were infected with Simliki Forest virus, and high-pressure frozen/freeze-substituted without chemical fixation. Post-stained plastic sections 0.25 and 0.12 (m in thickness were examined using the IVEM. The thicker sections were used to make a double-tilt tomographic reconstruction of a number of budding virus particles. The steps in the budding process could be clearly seen, including flattening of the virus particles as they reached the cell plasma membrane, and the details of formation of the envelope. A surface-rendered model was made using Sterecon for segmentation. The thinner sections were used for high-magnification tomographic reconstructions of the core particles. Core particles have not been extracted from cells for cryo-EM, so they are being studied in-situ. About 20 core particles were reconstructed from two double-tilt series. According to criteria we have been using for estimating tomographic resolution (phase residuals between neighboring z-slices), the resolution was about 2nm, based on a pixel size of 0.5nm. We are comparing this to the core particles inside complete virus particles reconstructed using averaging techniques after cryo-EM. Restoration techniques are being used to optimize the reconstructions, then averaging techniques will be applied in combination with the tomographic data. An abstract was written for the Scandinavian EM society meeting in Helsinki: Kan, S.T., Marko, M, Hultenby, K., Forsell, K., Garoff, H., Cheng, R.H. (1998) Structural stability of surface envelope and nucleocapsid core of alphaviruses. Proc. 50th Ann. Meet Scandinavian Soc. for Electron Microscopy E.L Punnonen and E. Heikinheimo, Eds. pp. 97-98. (