HPV CAPSID ASSEMBLY

流行病学和分子生物学研究都表明， 涉及人乳头瘤病毒类型，特别是HPV 16和HPV 18 宫颈鳞状细胞癌的病因学研究。 预防 HPV感染的疫苗，或使用疫苗或抗病毒药物 调节感染和肿瘤发展的药物是一种非常有效的方法。 重要目标。 我们实验室最近发展了一种 重组牛痘病毒表达系统，其允许组装 HPV蛋白进入衣壳提供了研究衣壳的机会 结构和组装。 这些信息可以用来设计 肽或抗体干扰感染性 病毒粒子 该研究将检查三种HPV类型：1，6和16， 评估乳头状瘤病毒之间共有的特征， 那些对特定类型来说是唯一的。 这项研究将联合收割机 生物化学、遗传学、微观和结构方法， 表征HPV衣壳。 这项建议的具体目标是：（1） 表征L1衣壳和衣壳的结构。 分子 将使用建模来确定是否存在结构相似性 乳头状瘤病毒的L1蛋白和 多瘤病毒。 冷冻电子显微照片和质量的图像分析 将使用扫描透射电子显微镜测量 在3.0 nm水平上研究L1衣壳和衣壳， 尝试定位L2。 将尝试使L1结晶 壳粒 (2)为了表征衣壳蛋白的结构域， 是自组装所必需的，包括fiv 含L1的亚基，L1之间的壳粒间接触 蛋白质在衣壳壳，和L1和L2之间的相互作用。 的 将评估L1形成壳体内和壳体内接触的能力 通过免疫共沉淀使用野生型和突变的衣壳蛋白， 分馏分析，电子显微镜，和双杂交系统 菲尔兹和宋 这两个混合动力系统也将用于识别 L1和L2之间的联系。 (3)为了表征翻译后 L1和L2蛋白的修饰。 翻译后 修饰将通过代谢标记来确定。 二维凝胶分析 和诱变。 将比较三种HPV之间的修饰 在牛痘和杆状病毒表达系统中寻找 一致的变化。 L2影响翻译后的能力 将检查L1的修改。 (4)表征 对病毒DNA的抑制作用的要求。 病毒DNA将 通过E1的表达瞬时复制到高拷贝数 和E2基因，或者将作为高拷贝附加体DNA存在于细胞系中，和 将使用重组牛痘病毒进行包装， 衣壳蛋白 将确定对L2蛋白的需求。

Both epidemiologic and molecular biological studies indicate that certai human papillomavirus types, particularly HPV 16 and HPV 18 are involved in the etiology of squamous cell carcinomas of the cervix. Prevention of HPV infection with vaccines, or the use of vaccines or antiviral agents to modulate infection and tumor development is an extremely important goal. The recent development in our laboratory of a recombinant vaccinia virus expression system that allows the assembly of HPV proteins into capsids provides an opportunity to study capsid structure and assembly. This information could be used to design peptides or antibodies that interfere with the formation of infectious virions. The study will examine three HPV types: 1,6 and 16 to allow an assessment of the features that are shared among papillomaviruses and those that are unique to a particular type. The study will combine biochemical, genetic, microscopic and structural approaches to characterize HPV capsids. The specific aims of this proposal are: (1) To characterize the structure of the L1 capsomers and capsid. Molecular modeling will be used to determine if there is structural similarity between the L1 proteins of papillomaviruses and the VP1 proteins of polyomaviruses. Image analysis of cryoelectron micrographs and mass measurements of scanning transmission electron micrographs will be used to study the L1 capsomers and the capsid at the 3.0 nm level and to attempt to locate L2. Attempts will be made to crystallize the L1 capsomer. (2) To characterize the domains of the capsid proteins that are required for self-assembly including the intracapsomer among the fiv L1 - containing subunits, the intercapsomer contacts among the L1 proteins in the capsid shell, and interactions between L1 and L2. The ability of L1 to form intra- and intercapsomer contacts will be assessed using wild type and mutated capsid proteins by coimmunoprecipitation, fractionation analysis, electron microscopy, and the two hybrid system of Fields and Song. The two hybrid system will also be used to identify contacts between L1 and L2. (3) To characterize post-translational modifications of the L1 and L2 proteins. Post-translational modifications will be determined by metabolic labeling. 2D gel analysis and mutagenesis. Modifications will be compared among the three HPV types in vaccinia and baculovirus expression systems to look for consistent changes. The ability of L2 to effect posttranslational modification of L1 will be examined. (4) To characterize the requirements for encapsidation of viral DNA. Viral DNA will be replicated to high copy number transiently by the expression of the E1 and E2 genes, or will exist as high copy episomal DNA in cell lines, and will be packaged using the recombinant vaccinia viruses to provide the capsid proteins. The requirement for the L2 protein will be determined.