MECHANISMS IN PAPOVAVIRUS ASSEMBLY

病毒的原子分辨率结构提供了一种新的 研究病毒与其细胞相互作用的框架 受体，设计抗病毒疗法，并了解 在复杂的大分子组装中的相互作用。 虽然这些 结构代表了很好的解决最终产品，病毒的过程 大会在其内部的管理方面仍然不明确， 病毒感染的细胞和病毒结构蛋白的性质 它们决定了组装的路径。 病毒组装的研究 设计抗病毒疗法的直接影响，但也有 与蛋白质折叠等一般生物学问题的相关性， 细胞内的运输，以及真核染色体与 结构蛋白 本建议的目的是为了了解结构， 乳多空病毒在原子、生物化学和细胞生物学上的组装 程度. 人乳头瘤病毒11亚型的结构分析 L1衣壳蛋白将使用重组L1蛋白作为载体进行研究。 乳多空病毒“次要”衣壳蛋白之间相互作用的底物 衣壳将使用病毒样的冷冻电子显微镜进行研究 在昆虫中表达多瘤病毒衣壳蛋白后纯化的颗粒 多瘤病毒VP 1/VP 2/VP 3或HPV的生物化学表征 L1/L2复合体。 使用突变型多瘤病毒VP 1蛋白的VP 1亚结构域 将分析羧基末端对大小的影响， 形状和曲率的衣壳在体外形成。 多瘤的相互作用 将分析具有细胞伴侣蛋白的VP 1，以确定是否 伴侣蛋白结合影响VP 1衣壳与衣壳的结合。 钙、蛋白质浓度和伴侣结合的体内效应 将使用表达于大肠杆菌中的突变体VP 1蛋白测试衣壳组装。 昆虫细胞

DESCRIPTION: The atomic resolution structures of viruses provide a new framework for studying the interaction of viruses with their cellular receptors, designing anti-viral therapeutics, and understanding the bonding interactions in complex macromolecular assemblies. Although these structures represent well-resolved end-products, the process of virus assembly remains ill-defined with regard to its regulation within virus-infected cells and the properties of the viral structural proteins which determine the pathways of assembly. The study of virus assembly has direct implications for designing anti-viral therapies, but also has relevance to the general biologic problems of protein folding, protein trafficking within cells, and the interaction of eukaryotic chromosomes with structural proteins. The aims of this proposal are directed at understanding the structure and assembly of papovaviruses at the atomic, biochemical, and cell biological levels. A structural analysis of the human papillomavirus (HPV) subtype-11 L1 capsid protein will be undertaken using a recombinant L1 protein as a substrate for interactions between the papovavirus "minor" capsid proteins and the capsid will be studied using cryoelectron microscopy of virus-like particles purified after expression of polyoma capsid proteins in insect cells, and biochemical characterization of polyomavirus VP1/VP2/VP3 or HPV L1/L2 complexes. Using mutant polyoma VP1 proteins subdomains of the VP1 carboxy terminus will be analyzed in relation to their effect upon the size, shape, and curvature of capsids formed in vitro. The interaction of polyoma VP1 with cell chaperone proteins will be analyzed to determine whether chaperone binding influences association of VP1 capsomeres into capsids. The in vivo effects of calcium, protein concentration, and chaperone binding on capsid assembly will be tested using mutant VP1 proteins expressed in insect cells.