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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 支持： （全部到瑞典Huddinge的Karolinska研究所的R. Holland Cheng；加利福尼亚大学的戴维斯大学） 欧盟委员会大分子蛋白质组学的框架计划，悬浮测试方法，2004-2006 VR-医学研究委员会的病毒构象与宿主入境有关，2002- 2004年 Wallenberg基金会表征α-2005年α-2005年细胞进入功能的激活机制 生物技术中心：病毒组装的结构功能研究，1997- 2004年 国际研究和高等教育基金会冷冻微镜和大分子复合物的3D图像重建，2002-2006 FFB知识改进基金会在病毒结构中的亚稳定性原理，2001- 2004年 早期病毒宿主细胞相互作用（待处理）的人类边界科学计划的结构和细胞生物学。 NIH待定赠款：衣壳组件和细胞相互作用中的急性肝炎病毒A和E 抽象的 仅通过检查整个安装座或厚部分才能获得原位病毒颗粒的3-D结构，以避免截断许多颗粒。原位颗粒需要与我们使用的平均方法具有玻璃体透明的分离病毒颗粒进行研究的颗粒相媲美。在先前的工作中，我们与RVBC合作，从经过高压冻结，冷冻取代并嵌入树脂中的感染组织培养细胞的部分进行断层造影重建。最初的结果非常令人鼓舞（Cheng等，2001; Sedzik等，2001），我们继续研究突变菌株的感染，α病毒生命周期中1型和2型细胞病蔬菜的作用以及其他病毒/宿主。但是，我们经常难以获得有关宿主细胞膜上病毒蛋白识别和组织病毒蛋白的令人信服的数据，这是回答重要问题的关键要求。 我们认为，现在在RVBC上可用的技术用于玻璃体冻结感染的细胞的电子断层扫描，这些细胞被没有固定，未含水和未染色的细胞，将提供我们在工作中需要进行的蛋白质结构的最佳保存。 大分子的结构及其周围环境的性质最终决定了功能。这是病毒的结构蛋白，不仅是将颗粒保持在一起的原因，而且还负责触发病毒组装过程中构象变化以成功地传递基因组（Cheng等，1995; Smith等，1995）。这需要特定理解病毒颗粒中内置的灵活性，以适应通过宿主细胞的各种构象变化的需求（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新的分辨率。获得的结果对于调节我们的病毒组装和进入机制的概念将非常重要。项目中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Semliki Forest病毒（SFV）的野生型和突变菌株，以评估核Ocapsid的构象变化，因为它在细胞质中是新形成的，因为它在成熟的细胞外粒子中被萌芽过程包围，并最终将其遗传含量传递到新的细胞中。我们对在发芽过程中的信封层及其过渡特别感兴趣。最终目标是了解尖峰尖峰相互作用如何迫使外部磷脂层形成毛孔，以及在此事件中如何锚定峰值。 细胞内的核Ocapsids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的核蛋白质，因为由于没有峰值而阻止了萌芽（Suomalainen等，1992）。在萌芽过程中，将使用野生型SFV进行研究。一个替代的组装过程，其中病毒颗粒在某些突变体中的细胞膜或附近（Forsell等，1996）也将进行研究。发现的各种颗粒的t数应该有助于我们了解基因组的大小如何影响α病毒形成的表面晶格。新信息将与通过平均方法获得的完整病毒颗粒的高分辨率结构集成（Cheng等，1995； Haag等，2002）。我们计划将电子断层扫描和傅立叶平均技术相结合，以获得原位核ocapsid颗粒的高分辨率结构。 在两种制备方法之后，将检查感染的BHK组织培养细胞（Kan等，1998）：（1）在冷冻水合状态下对整个细胞进行冻结并检查细胞外围细胞，以及（2）高压细胞和切割冷冻冻结的水分的高压力。第一种方法可能对研究病毒生命周期中涉及的细胞中膜系统的组织以及细胞膜上出现病毒颗粒的分布模式有用。第二种方法应使我们能够检查层次图中的薄片并定位和识别病毒蛋白。 可以通过比较从完整细胞表面释放的完整病毒颗粒的层析成像重建可以清楚地评估成功，并具有先前使用傅立叶平均技术制成的隔离颗粒的高分辨率重建（Baker和Cheng，1996; Cheng等，1992; 1992; 1994; 1994; Fuller等，1996）。我们还希望利用基序搜索（Rath等，2004）来定位病毒蛋白。 我们非常高兴的是，RVBC提出的提案提出了对合作者使用的冷冻水分层析成像技术（Hsieh等，2002），因为我们认为这是获取所需信息的关键。在奥尔巴尼400kV EM中添加成像能过滤器还将使我们能够获得整体，螺旋形的，感染的细胞的高质量断层扫描。当我们努力争取更高的分辨率时，原位和孤立的病毒颗粒之间的比较将为评估层析成像重建质量提供一种方法。 参考 1。Baker，T.S。和Cheng，R.H。（1996）一种基于模型的方法，用于确定低温电子显微镜成像的生物学大分子的取向。 J结构生物。 116（1）：120 130。 2。Cheng，R.H.，Kuhn，R.J.，Olson，N.H.，Rossmann，M.G.，Choi，H.K.，Smith，T.J。和贝克，T.S。 （1995）在包膜病毒中的核蛋白质和糖蛋白组织。单元80（4）：621 630。 3。Cheng，R.H.，N.H。Olson和T.S. Baker （1992）花椰菜马赛克病毒：420个亚基（t = 7），多层结构。病毒学186（2）：655 668。 4。Cheng，R。H.，Reddy，V。S.，Olson，N。H.，Fisher，A。J.，Baker，T。S.和Johnson，J。E.（1994）t = 3 t = 3 Icosahedral Animal Virus的功能含义由冷冻电子显微镜和X射线晶体学建立。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年）（2001）将高分辨率和低分辨率数据汇总在一起：萌芽的alpheped Alphavirus。显微镜。微分析。 7（补充2）：104-105。 6。 Embo J. 15（23）：6495 6505。 7。Fuller，S.D。，Butcher，S.J.，Cheng，R.H。和Baker，T.S。 （1996）二十面体颗粒的三维重建罕见线。 J. struct。生物。 116（1）：48 55。 8。Garoff，H，Sj¿Berg，M和Cheng，R。H.（2004）。 α病毒的萌芽。病毒res。在印刷中。 9。Garoff，H。和Cheng，R。H.（2001）。信封形成与α病毒融合之间的缺失联系。趋势微生物，9：408-410。 10。Gibbons，dl，A AHN，M Liao，L Hammar，Rh Cheng和M Kielian（2004）融合肽与Semliki Forest病毒的膜插入膜插入中的多步调节。 J Virol，78：3312-3318 11。Haag，L.，Garoff，H.，Xing，L.，Hammar，L.，Kan，S，S和Cheng，R。（2002）α的酸蛋白壳中酸蛋白壳的运动将尖刺转化为膜融合模式。 Embo J，21：4402-4410。 12。Hammar，L，Markarian，S，Haag，L，Lankinen，H，Salmi，A和Cheng，RH（2003）灌注重排，导致Semliki Forest病毒的融合肽暴露。 J Biol Chem，278：7189-98。 13。Hsieh，C.，Marko，M.，Frank，J。和Mannella，C.A。（2002）对冷冻水合组织切片的电子断层扫描分析。 J. struct。生物。 138：63-73。 14。Kan，S.，Marko，M.，Hultenby，K.，Forsell，K.，Garoff，H。和Cheng，R。（1998年）表面膜的结构稳定性和α的核Ocapsid核心。 Proc。扫描。 Soc。电子。显微镜。 50:97 98。 15。Rath，B.K.，Hegerl，R.，Leith，A.，Shaikh，T.R.，Wagenknecht，T。和Frank，J。（2004）使用局部标准化的交叉相关函数快速3D图案搜索EM密度图。 J. struct。 Biol。，145：84-90。 16。Sedzik，J.，Hammar，L.，Haag，L.，Skoging-nyberg，U.，Tars，K.，Marko，M。和Cheng，R。H.（2001）包裹病毒的结构蛋白质组学：结晶，晶体学，诱变，诱变和低温 - 电子显微镜。最近的Res Devel Virol 3：41-60 17。Smith，T.J.，Cheng，R.H.，Olson，N.H.，Peterson，P.，Chase，E.，Kuhn，R.J。和贝克，T.S。 （1995）通过低温电子显微镜观察到的α病毒上的假定受体结合位点。 Proc。纳特。学院。科学。美国92（23）：10648 10652。 18。Suomalainen，M.，Liljestrom，P。和Garoff，H。（1992）尖峰蛋白核ocapsid相互作用驱动α病毒的萌芽。 J. Virol。 66（8）：4737 4747。