This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.SUPPORT: (All to R. Holland Cheng, Karolinska Institute, Huddinge, Sweden; Univ. of Calif. at Davis)Framework Programme of European Commission Macromolecule Proteomics by Levitation Test Method , 2004-2006VR - Medical Research Council Viral Conformations Relevant to Host Entry, 2002-2004Wallenberg Foundation Characterization of the Activation Mechanism for Cell-Entry Functions in Alphavirus, 2004-2005Centrum for Biotechnology: Structure-Function Study of Virus Assembly, 1997-2004STINT Foundation for International Research and Higher Education Cryo-microscopy and 3D Image Reconstruction of Macromolecular Complexes, 2002-2006FFB Foundation for Knowledge Improvement Probing Metastability Principles in Virus Structures, 2001-2004Human Frontier Scientific Program Structural and Cellular Biology of Early Virus-Host Cell Interaction (pending). NIH pending grant: Acute Hepatitis Virus A and E in Capsid Assembly and Cell InteractionsABSTRACTThe 3-D structure of in-situ virus particles can only be obtained by examining whole mounts or thick sections, in order to avoid truncation of many particles. The in-situ particles need to be comparable to those we have studied using averaging methods with vitreously-frozen isolated virus particles. In previous work, we collaborated with the RVBC to make tomographic reconstructions from sections of infected tissue-culture cells that had been high-pressure frozen, freeze-substituted and embedded in resin. Initial results were very encouraging (Cheng, et al., 2001; Sedzik et al., 2001), and we went on to study infection of mutant strains, the role of cytopathic vesicles type 1 and 2 in the alphavirus life cycle, and other virus/host pairs. However, we frequently had difficulty in obtaining convincing data regarding identification and organization of viral proteins on the host cell membrane, a critical requirement for answering important questions. We believe that the techniques now available at the RVBC for electron tomography of vitreously-frozen infected cells that are unfixed, undehydrated, and unstained will provide the optimal preservation of protein structure that we need to go forward in our work.The structure of a macromolecule and the properties of its surroundings ultimately dictate function. Thus, the structural proteins of viruses are responsible not only for keeping the particle together, but also for triggering conformational changes during the virus assembly to successfully deliver the genome (Cheng et al., 1995; Smith et al., 1995). This requires specific understanding of the built in flexibility in viral particles to accommodate various needs of conformational changes through the host cell (Hammar et al., 2003; Garoff and Cheng, 2001; Gibbons et al., 2004; Garoff et al., 2004). This project will bring our understanding of viral infection to a new level of resolution. The results obtained will be extremely important in modulating our conception of virus assembly and entry mechanisms in general. The method developed in the project will be useful for analysis of other virus systems as well as large enzyme complexes.Both wild type and mutant strains of Semliki Forest virus (SFV) will be studied to assess the conformational changes in the nucleocapsid as it is newly-formed in the cytoplasm, as it is surrounded by an envelope during budding, as a mature extracellular particle, and finally as it delivers its genetic content into a new cell. We are especially interested in the envelope layer and its transitions during the budding process. An ultimate goal would be to understand how spike spike interaction forces the external phospholipid layer to form pores, and how the spikes are anchored during this event. The intracellular nucleocapsids will be studied using a spike variant of SFV. This variant produces large amounts of nucleocapsids in the cytoplasm because budding is prevented since no spikes are made (Suomalainen, et al., 1992). The process of budding and the formation of the envelope during budding will be studied using wild-type SFV. An alternative assembly process, in which the virus particles assemble at or near the cell membrane in some mutants (Forsell, et al., 1996) will also be studied. The T number of the various particles found should help us understand how the size of the genome influences the surface lattice formed by alphaviruses. The new information will be integrated with the high resolution structure of intact virus particles obtained by averaging methods (Cheng et al., 1995; Haag et al., 2002). We plan to combine electron tomography and Fourier averaging techniques to obtain a high-resolution structure of in situ nucleocapsid particles. Infected BHK tissue culture cells (Kan et al., 1998) will be examined cryo-electron tomograms after two methods of preparation: (1) plunge freezing whole cells and examination of the cell periphery in the frozen hydrated state, and (2) high pressure freezing pelleted cells and cutting frozen-hydrated sections. The first method is likely to be useful for study of the organization of membrane systems in the cell that are involved in the viral life cycle, and for distribution patterns of budding virus particles on the cell membrane. The second method should enable us to examine thin slices from tomograms and locate and identify viral proteins. Success can be clearly evaluated by comparing tomographic reconstructions of complete virus particles releasing from the surface of intact cells with high resolution reconstructions of isolated particles previously made using Fourier averaging techniques (Baker and Cheng, 1996; Cheng, et al., 1992; 1994; Fuller et al., 1996). We also hope to take advantage of motif-searching (Rath, et al., 2004) to computationally locate viral proteins.We are very pleased that the RVBC proposes to make the technique of electron tomography of frozen-hydrated sections (Hsieh, et al., 2002) available to collaborators, as we feel that this is the key to obtaining the information we require. The addition of the imaging energy filter to the Albany 400kV EM will also allow us to obtain high-quality tomograms of the edges of whole, plunge-frozen, infected cells. The comparison between in-situ and isolated virus particles will provide a means for evaluating quality of tomographic reconstructions, as we strive for ever higher resolution.References1. Baker, T.S. and Cheng, R.H. (1996) A model based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy. J Struct Biol. 116(1):120 130.2. Cheng, R.H., Kuhn, R.J., Olson, N.H., Rossmann, M.G., Choi, H.K., Smith, T.J. and Baker, T.S. (1995) Nucleocapsid and glycoprotein organization in an enveloped virus. Cell 80(4):621 630.3. Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure. Virology 186(2):655 668.4. Cheng, R. H., Reddy, V.S., Olson, N.H., Fisher, A.J., Baker, T.S. and Johnson, J.E. (1994) Functional implications of quasi equivalence in a T = 3 icosahedral animal virus established by cryo electron microscopy and X ray crystallography. Structure 2(4):271 282.5. Cheng, R.H., Hultenby, K., Haag, L., Forsell, K., Garoff, H., Hsieh, C.-E., and Marko, M. (2001) Bringing together high- and low-resolution data: elecron tomography of budding enveloped alphavirus. Microsc. Microanal. 7(Suppl. 2):104-105.6. Forsell, K., Griffiths, G. and Garoff, H. (1996) Preformed cytoplasmic nucleocapsids are not necessary for alphavirus budding. EMBO J. 15(23):6495 6505.7. Fuller, S.D., Butcher, S.J., Cheng, R.H. and Baker, T.S. (1996) Three dimensional reconstruction of icosahedral particles the uncommon line. J. Struct. Biol. 116(1):48 55.8. Garoff, H, Sj berg, M and Cheng, RH (2004). Budding of alphaviruses. Virus Res. in press. 9. Garoff, H., and Cheng, R. H. (2001). The missing link between envelope formation and fusion in alphaviruses. Trend Microbiol, 9:408-410. 10. Gibbons, DL, A Ahn, M Liao, L Hammar, RH Cheng, and M Kielian (2004) Multistep regulation in membrane insertion of the fusion peptide with Semliki Forest virus. J Virol, 78: 3312-331811. Haag, L., Garoff, H., Xing, L., Hammar, L., Kan, S, and Cheng, RH (2002) Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode. EMBO J, 21:4402-4410. 12. Hammar, L, Markarian, S, Haag, L, Lankinen, H, Salmi, A, and Cheng, RH (2003) Prefusion rearrangements resulting in fusion peptide exposure in Semliki Forest virus. J Biol Chem, 278:7189-98. 13. Hsieh, C, Marko, M., Frank, J., and Mannella, C.A. (2002) Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol. 138:63-73.14. Kan, S., Marko, M., Hultenby, K., Forsell, K., Garoff, H. and Cheng, R. (1998) Structural stability of surface envelope and nucleocapsid core of alphaviruses. Proc. Scand. Soc. Elec. Microsc. 50:97 98.15. Rath, B.K., Hegerl, R., Leith, A., Shaikh, T.R., Wagenknecht, T., and Frank, J. (2004) Fast 3D motif search of EM density maps using a locally normalized cross-correlation function. J. Struct. Biol., 145:84-90.16. Sedzik, J., Hammar, L., Haag, L., Skoging-Nyberg, U., Tars, K., Marko, M., and Cheng, R. H. (2001) Structural proteomics of enveloped viruses: Crystallization, crystallography, mutagenesis and cryo-electron microscopy. Recent Res Devel Virol 3: 41-6017. Smith, T.J., Cheng, R.H., Olson, N.H., Peterson, P., Chase, E., Kuhn, R.J. and Baker, T.S. (1995) Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy. Proc. Natl. Acad. Sci. USA 92(23):10648 10652.18. Suomalainen, M., Liljestrom, P. and Garoff, H. (1992) Spike protein nucleocapsid interactions drive the budding of alphaviruses. J. Virol. 66(8):4737 4747.

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