Investigation of early events in oncogenic HPV infection

描述（由申请人提供）：本提案的目的是研究和比较致癌HPV 16、18和31生命周期中的早期事件。具体来说，我们希望确定用于宿主细胞渗透的精确内吞途径，并确定细胞进入过程所需和诱导的细胞信号传导事件类型。癌症生物学的固有差异以及这些致癌HPV的衣壳组成保证了这些病毒-宿主细胞相互作用的研究。我们将通过感染性/中和时程实验以及通过共聚焦和电子显微镜直接可视化来确定摄取的动力学。将通过药理学抑制实验和病毒体与特异性内吞标志物之间共定位的直接可视化的组合来确定特异性内吞进入途径。响应于病毒体的内吞摄取的信号转导将通过磷蛋白的上调来评估，其身份可以通过质谱来揭示。还将确定特定激酶和磷酸酶活性的药理学抑制对HPV细胞内运输和感染性的影响。总之，这些研究的结果将有助于定义最初HPV-宿主细胞相互作用期间的分子事件，并将为更详细地研究这些关键过程（胞质易位，病毒体解体等）的细节奠定基础。

DESCRIPTION (provided by applicant): The goal of this proposal is to study and compare the early events in the life cycle of oncogenic HPV16, 18, and 31. Specifically we want to identify the precise endocytic pathways used for host cell penetration and identify what types of cellular signalling events are both required for and induced by the process of cell entry. Inherent differences in the cancer biology as well the capsid composition of these oncogenic HPVs warrants the investigation of these viral-host cell interactions. We will determine the kinetics of uptake by both infectivity/neutralization timecourse experiments as well as by direct visualization by confocal and electron microscopy. The specific endocytic routes of entry will be determined through a combination of pharmacological inhibition experiments and direct visualization of colocalization between virions and specific endocytic markers. Signal transduction in response to the endocytic uptake of virions will be assessed by the upregulation of phospho-proteins, the identity of which can be revealed by mass spectroscopy. The effects of pharmacological inhibition of specific kinase and phosphatase activities on HPV intracellular trafficking and infectivity will also be determined. Taken together, results from these studies will help define the molecular events during the initial HPV-host cell interactions and will set the stage for more detailed investigation into specifics of these critical processes (cytosolic translocation, virion disassembly, etc.)