HPV CAPSID ASSEMBLY

流行病学和分子生物学研究都表明， 人乳头瘤病毒类型，特别是HPV16和HPV18型 在宫颈鳞状细胞癌的病因学中。预防 使用疫苗或使用疫苗或抗病毒药物感染HPV的情况 调节感染和肿瘤发展的药物是一种极端 重要的目标。我们实验室最近的一项研究进展 重组痘苗病毒表达系统允许组装 HPV蛋白进入衣壳为研究衣壳提供了机会 结构和装配。这些信息可以用来设计 干扰感染性病毒形成的多肽或抗体 病毒粒子。这项研究将检查三种HPV类型：1、6和16，以允许 对乳头状瘤病毒和 它们是特定类型所特有的。这项研究将结合 生化、遗传、微观和结构方法 描述HPV衣壳的特征。这项建议的具体目的是：(1) 对L1囊膜和衣壳的结构进行表征。分子 将使用建模来确定是否存在结构相似性 乳头瘤病毒的L1蛋白和VP1蛋白之间的关系 多瘤病毒。低温电子显微照片和质量的图像分析 将使用扫描电子显微镜的测量方法 在3.0 nm水平上研究L1囊膜和衣壳 尝试找到L2。将尝试使L1具体化 胶囊剂。(2)鉴定衣壳蛋白的结构域 是自组装所必需的，包括五个中的内囊体 L1-含有亚基，包膜间相互接触 衣壳中的蛋白质，以及L1和L2之间的相互作用。这个 将评估L1形成囊内和囊间接触的能力 使用免疫共沉淀的野生型和突变衣壳蛋白， 分馏分析、电子显微镜和双杂化系统 菲尔兹和宋。这两个混合系统还将用于识别 L1和L2之间的接触。(3)后翻译的特征 L1和L2蛋白的修饰。翻译后 修饰将通过新陈代谢标签来确定。二维凝胶分析 和诱变。将对三种HPV的修改进行比较 痘苗病毒和杆状病毒表达系统中要寻找的类型 始终如一的变化。第二语言影响翻译后翻译的能力 将研究L1的修改。(4)描述 病毒DNA的封装要求。病毒DNA将会是 通过E1的表达瞬时复制到高拷贝数 和E2基因，或将在细胞系中以高复制异构体DNA的形式存在，以及 将使用重组痘苗病毒进行包装，以提供 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.