The role of virus structure and host cell membrane trafficking as well as signalling factors in Human Papillomavirus Type 16 endocytosis

宫颈癌是由人乳头瘤病毒（HPV）引起的一种恶性肿瘤。在宫颈癌中发现的主要HPV亚型是HPV-16。除了通过内吞作用发生外，对HPV进入宿主细胞知之甚少。我们的研究结果表明，HPV-16进入宿主细胞的网格蛋白和小窝蛋白的独立机制，功能的一些，但不是所有的特点，巨胞饮，这可能是一种新的内吞途径进入细胞。我们建议进一步研究HPV-16通过内吞作用进入细胞。该项目有以下目的：-确定参与HPV-16内吞作用的大多数宿主细胞因子-阐明宿主细胞因子和信号传导在HPV-16内吞作用动力学中的作用-了解硫酸化碳水化合物在HPV-16结合中的分子作用-确定HPV-16病毒粒子中作为内吞进入功能“线索”的结构变化 主要应用的方法包括各种活细胞显微镜技术以及细胞生物学、形态学、遗传学和生物化学技术。为了对HPV进入的要求进行分类，将在自动化高通量测定中进行全基因组siRNA介导的基因沉默筛选。为了获得HPV内吞事件的时间和空间信息，将使用几种先进的光学显微镜技术（宽视野，共聚焦和全内反射显微镜）来跟踪单个HPV及其与活细胞内吞细胞器特异性标志物的关联。为了确定最特异性地用于HPV-16与宿主细胞结合的碳水化合物，我们将筛选碳水化合物微阵列。为了阐明内吞过程中病毒颗粒的结构变化，我们将对感染细胞中的病毒颗粒进行分离、生化和超微结构分析。这些结果将有助于理解HPV进入宿主细胞的过程，并有助于分析哺乳动物细胞内吞的运输。

Several human papillomaviruses (HPV) are the central cause for cervical cancer. The major HPV subtype found in cervix carcinomas is HPV-16. Little is known about the entry of HPVs into host cells except that it occurs via endocytosis. Our results indicate that HPV-16 enters host cells by a clathrin- and caveolin-independent mechanism that features some but not all characteristics of macropinocytosis, and that likely represents a novel endocytic route into cells. We propose to further investigate the cellular entry of HPV-16 by endocytosis. This project has the following aims: - To identify most of the host cell factors involved in HPV-16 endocytosis- To elucidate the role of host cell factors and signalling in the dynamics of HPV-16 endocytosis- To understand the molecular role of sulfated carbohydrates in binding of HPV-16- To identify structural changes in HPV-16 virions that serve as functional ‚cues’ for endocytic entry and trafficking as well as for uncoating of the virus.The main methods to be applied include various techniques for live cell microscopy as well as cell biological, morhological, genetic, and biochemical techniques. To categorise the requirements for HPV entry a genome-wide siRNA-mediated gene silencing screen will be performed in an automated high through-put assay. To gain temporal and spatial information on the events in HPV endocytosis, several advanced light microscopic techniques (wide field, confocal, and total internal reflection microscopy) will be used to follow single HPVs and their association with specific markers for endocytic organelles in living cells. To determine the carbohydrates that serve most specifically for binding of HPV-16 to host cells we will screen a carbohydrate microarrays. To elucidate structural changes in virus particles during endocytosis we will use isolation, biochemical, and ultrastructural analysis of virus particles from infected cells. The results are expected to contribute both to the understanding of HPV entry into host cells, and to the analysis of endocytic traffic in mammalian cells.