IN-SITU STUDY OF BUDDING AND ASSEMBLY OF SEMLIKI FOREST VIRUS PARTICLES

SEMLIKI 森林病毒颗粒出芽和组装的原位研究

• 批准号: 7598345
• 负责人: R.Holland Cheng
• 金额: $ 0.23万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598345
• 关键词: Acids; Acute Hepatitis; Alphavirus; Animal Viruses; Binding Sites; Biological; Biological Preservation; Biotechnology; Capsid; Cauliflower Mosaic Virus; Cell Communication; Cell membrane; Cells; Cellular biology; Computer Retrieval of Information on Scientific Projects Database; Conceptions; Cryoelectron Microscopy; Crystallization; Crystallography; Cytoplasm; DNA Sequence Rearrangement; Data; Education; Electrons; European; Event; Foundations; Freezing; Funding; Genetic; Genome; Glycoproteins; Goals; Grant; Hepatitis Viruses; Human; Hylobates Genus; Image; In Situ; Infection; Institutes; Institution; International; Knowledge; Life Cycle Stages; Link; Macromolecular Complexes; Maps; Medical Research; Membrane; Membrane Fusion; Methods; Microscopy; Modeling; Molecular Conformation; Movement; Multienzyme Complexes; Mutagenesis; N(delta)-acetylornithine, -isomer; N-dodecanoylglutamic acid, -isomer, sodium salt; Nucleocapsid; Nucleocapsid Proteins; Numbers; Pattern; Peptides; Phospholipids; Plant Resins; Pliability; Preparation; Process; Property; Proteomics; Regulation; Research; Research Personnel; Resolution; Resources; Role; Semliki forest virus; Slice; Source; Structural Protein; Structure; Surface; Sweden; System; Tars; Techniques; Testing; Thick; Three-Dimensional Image; Three-Dimensional Imaging; Tissues; Tomogram; United States National Institutes of Health; Variant; Vesicle; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Work; X-Ray Crystallography; abstracting; alpha-difluoromethyl-DOPA, -isomer; alpha-methylornithine dihydrochloride, -isomer; base; density; electron tomography; extracellular; frontier; interest; macromolecule; mutant; particle; pressure; prevent; programs; protein structure; receptor binding; reconstruction; size; success; three dimensional structure; tissue/cell culture; tomography; trend; virology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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 , 2004-2006 VR - Medical Research Council Viral Conformations Relevant to Host Entry, 2002-2004 Wallenberg Foundation Characterization of the Activation Mechanism for Cell-Entry Functions in Alphavirus, 2004-2005 Centrum for Biotechnology: Structure-Function Study of Virus Assembly, 1997-2004 STINT Foundation for International Research and Higher Education Cryo-microscopy and 3D Image Reconstruction of Macromolecular Complexes, 2002-2006 FFB Foundation for Knowledge Improvement Probing Metastability Principles in Virus Structures, 2001-2004 Human 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 Interactions ABSTRACT The 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. References 1. 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: 3312-3318 11. 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: 41-60 17. 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 支持： （全部致 R. Holland Cheng，瑞典胡丁厄卡罗林斯卡学院；加州大学戴维斯分校） 2004-2006年欧盟委员会悬浮测试法高分子蛋白质组学框架计划 VR - 医学研究委员会与宿主进入相关的病毒构象，2002-2004 年 Wallenberg 基金会对甲病毒细胞进入功能激活机制的表征，2004-2005 年 生物技术中心：病毒组装的结构功能研究，1997-2004 STINT 国际研究和高等教育基金会 大分子复合物的冷冻显微镜和 3D 图像重建，2002-2006 FFB 知识改进基金会探索病毒结构的亚稳定性原理，2001-2004 年 人类前沿科学计划早期病毒-宿主细胞相互作用的结构和细胞生物学（待定）。 NIH 待拨款：衣壳组装和细胞相互作用中的急性甲型肝炎病毒和乙型肝炎病毒 抽象的 原位病毒颗粒的 3-D 结构只能通过检查整个样本或厚切片来获得，以避免许多颗粒被截断。原位病毒颗粒需要与我们使用平均方法研究的颗粒进行比较。在之前的工作中，我们与 RVBC 合作，对高压冷冻、冷冻替代并嵌入树脂中的受感染组织培养细胞切片进行断层扫描重建。结果非常令人鼓舞（Cheng 等，2001；Sedzik 等，2001），我们继续研究突变株的感染、1 型和 2 型细胞病变囊泡在甲病毒生命周期中的作用以及其他病毒然而，我们经常难以获得有关宿主细胞膜上病毒蛋白的识别和组织的令人信服的数据，这是回答重要问题的关键要求。 我们相信，RVBC 目前可用于对未固定、未脱水和未染色的玻璃体冷冻感染细胞进行电子断层扫描的技术将为我们工作中所需的蛋白质结构提供最佳保存。 大分子的结构及其周围环境的特性最终决定了功能，因此，病毒的结构蛋白不仅负责将颗粒保持在一起，而且还负责在病毒组装过程中触发构象变化，以成功传递基因组（Cheng等人）。 al.，1995；Smith 等，1995）这需要对病毒颗粒的内在灵活性有具体的了解，以适应宿主细胞构象变化的各种需求（Hammar 等，2003；Garoff 和Cheng，2001；Gibbons 等，2004；Garoff 等，2004）。 该项目将使我们对病毒感染的理解达到一个新的水平，所获得的结果对于调整我们对病毒组装和进入机制的概念非常重要。该项目开发的方法将有助于分析其他病毒。系统以及大型酶复合物。 将研究塞姆利基森林病毒（SFV）的野生型和突变株，以评估核衣壳的构象变化，因为它是在细胞质中新形成的，因为它在出芽期间被包膜包围，作为成熟的细胞外颗粒，最后，当它将其遗传内容传递到新细胞中时，我们对包膜层及其在出芽过程中的转变特别感兴趣，最终目标是了解尖峰相互作用如何迫使外部磷脂层发生变化。形成孔隙，以及在此事件期间尖峰如何锚定。 细胞内核衣壳将使用 SFV 的尖峰变体进行研究，该变体在细胞质中产生大量核衣壳，因为由于没有尖峰的形成而阻止了出芽（Suomalainen 等，1992）。出芽期间的包膜将使用野生型 SFV 进行研究。另一种组装过程是，病毒颗粒在某些突变体的细胞膜处或附近组装。 （Forsell 等人，1996）还将研究发现的各种颗粒的 T 数，这将有助于我们了解基因组的大小如何影响甲病毒形成的表面晶格。新的信息将与高分辨率的理解相结合。通过平均方法获得完整病毒颗粒的结构（Cheng等人，1995；Haag等人，2002）我们计划结合电子断层扫描和傅里叶平均技术来获得高分辨率的病毒颗粒结构。原位核衣壳颗粒。 受感染的 BHK 组织培养细胞（Kan 等人，1998）将在两种制备方法后进行冷冻电子断层扫描检查：（1）骤冷全细胞并检查冷冻水合状态的细胞外围，以及（2）高浓度压力冷冻沉淀细胞和切割冷冻水合切片可能有助于研究细胞中参与病毒生命周期的膜系统的组织以及出芽的分布模式。第二种方法应该使我们能够检查断层图像的薄片并定位和识别病毒蛋白。 通过将从完整细胞表面释放的完整病毒颗粒的断层扫描重建与先前使用傅里叶平均技术进行的分离颗粒的高分辨率重建进行比较，可以清楚地评估成功与否（Baker 和 Cheng，1996；Cheng 等人，1992；1994； Fuller 等人，1996）我们还希望利用基序搜索（Rath 等人，2004）来通过计算定位病毒蛋白。 我们非常高兴 RVBC 提议向合作者提供冷冻水合切片的电子断层扫描技术（Hsieh 等，2002），因为我们认为这是获得我们所需信息的关键。奥尔巴尼 400kV 电磁成像能量过滤器还将使我们能够获得整个冷冻感染细胞边缘的高质量断层图。原位病毒和分离病毒之间的比较。随着我们努力追求更高的分辨率，粒子将提供一种评估断层扫描重建质量的方法。 参考 1. Baker, T.S. 和 Cheng, R.H. (1996) 通过冷冻电子显微镜成像确定生物大分子方向的模型方法。 116(1):120 130。 2. Cheng, R.H.、Kuhn, R.J.、Olson, N.H.、Rossmann, M.G.、Choi, H.K.、Smith, T.J. 和 Baker, T.S. (1995) 包膜病毒中的核衣壳和糖蛋白组织 80(4):621 630。 3. Cheng, R.H.、Olson, N.H. 和 Baker, T.S. (1992) 花椰菜花叶病毒：420 个亚基 (T = 7)，多层结构。 4. Cheng, R. H.、Reddy, V.S.、Olson, N.H.、Fisher, A.J.、Baker, T.S. 和 Johnson, J.E. (1994) 通过冷冻电子显微镜和 X 射线晶体学建立的 T = 3 二十面体动物病毒中的准等效性的功能意义结构2(4)：271 282。 5. Cheng, R.H.、Hultenby, K.、Haag, L.、Forsell, K.、Garoff, H.、Hsieh, C.-E. 和 Marko, M. (2001) 结合高分辨率和低分辨率数据：出芽包膜甲病毒的电子断层扫描。Microanal。7（增刊2）：104-105。 6. Forsell, K.、Griffiths, G. 和 Garoff, H. (1996) 甲病毒出芽不需要预先形成的细胞质核衣壳。 15(23):6495 6505。 7. Fuller, S.D.、Butcher, S.J.、Cheng, R.H. 和 Baker, T.S. (1996) 二十面体粒子的三维重建，J. Biol。 8. 加洛夫，H，Sj¿ berg, M 和 Cheng, RH (2004) 病毒研究。 9. Garoff, H. 和 Cheng, R. H. (2001) 甲病毒中包膜形成和融合之间缺失的联系，9:408-410。 10. Gibbons、DL、A Ahn、M Liao、L Hammar、RH Cheng 和 M Kielian (2004) Semliki Forest 病毒融合肽膜插入的多步调节，78：3312-3318。 11. Haag, L.、Garoff, H.、Xing, L.、Hammar, L.、Kan, S 和 Cheng, RH (2002) 甲病毒糖蛋白壳中酸诱导的运动将刺突转变为膜融合EMBO J，21：4402-4410。 12. Hammar, L、Markarian, S、Haag, L、Lankinen, H、Salmi, A 和 Cheng, RH (2003) 融合肽在 Semliki Forest 病毒中暴露的预融合重排，278:7189-98。 。 13. Hsieh, C、Marko, M.、Frank, J. 和 Mannella, C.A.（冷冻水合组织切片的电子断层扫描分析）138：63-73。 14. Kan, S.、Marko, M.、Hultenby, K.、Forsell, K.、Garoff, H. 和 Cheng, R. (1998) 甲病毒的表面包膜和核衣壳核心的结构稳定性。电子。 50:97 98。 15. Rath, B.K.、Hegerl, R.、Leith, A.、Shaikh, T.R.、Wagenknecht, T. 和 Frank, J. (2004) 使用局部标准化互相关函数对 EM 密度图进行快速 3D 基序搜索。结构杂志，145：84-90。 16. Sedzik, J.、Hammar, L.、Haag, L.、Skoging-Nyberg, U.、Tars, K.、Marko, M. 和 Cheng, R. H. (2001) 有包膜病毒的结构蛋白质组学：结晶、晶体学、诱变和冷冻电子显微镜。最近的 Res Devel Virol 3：41-60。 17. Smith, T.J.、Cheng, R.H.、Olson, N.H.、Chase, E.、Kuhn, R.J. 和 Baker, T.S.（1995）通过冷冻电子显微镜观察的甲病毒上的假定受体结合位点。美国科学 92(23):10648 10652。 18. Suomalainen, M.、Liljestrom, P. 和 Garoff, H. (1992) 刺突蛋白核衣壳相互作用驱动甲病毒的出芽 66(8):4737 4747。