The Role of Caspase Activation in the Differentiation-Dependent Life Cycle of HPV

描述(申请人提供)：我的职业目标是成为DNA肿瘤病毒领域的一名领先的独立科学家。在独立之路奖的指导阶段，我将努力进一步建立我在HPV领域的出版记录，这将为我作为教师所做的研究奠定基础。这个项目将提供新技术(共聚焦显微镜、流式细胞术、凝胶偏移)的培训，这些技术将适用于我作为独立研究员进行的研究。Laimins博士以及微生物学-免疫学系的其他教员的专业知识对于学习成为一名成功的独立研究人员所需的技能将是非常宝贵的。 人乳头瘤病毒是一种小的DNA病毒，被认为是宫颈癌的病原体。HPV的感染生命周期依赖于细胞因子和上皮分化。分化触发了生命周期的生产阶段，包括病毒基因组扩增和病毒粒子产生。该项目的长期目标是了解调节依赖于分化的病毒事件激活的机制。我最近证明，HPV-31在分化时刺激低水平的caspase激活，这是线粒体凋亡途径的特征，也是通过切割病毒复制蛋白E1来扩增病毒基因组所必需的。根据E6和E7都能刺激caspase激活的发现，以及对几种HPV蛋白中caspase裂解位点的鉴定，我推测：HPV通过E6和E7诱导分化细胞中caspase的激活，从而改变病毒蛋白作为激活晚期病毒事件的机制的功能。在指导阶段的具体目标1中，我将通过检测E6和E7对线粒体膜完整性的影响以及确定E6和E7中激活caspase所必需的结构域，来确定HPV蛋白在分化细胞中激活caspase的机制(S)。在独立阶段启动的特定目标2中，我将通过体外和体内研究确定HPV蛋白的caspase裂解如何在生命周期中促进分化相关事件。阐明E6和E7在分化上皮过程中介导caspase激活从而导致病毒蛋白切割的途径将有助于深入了解HPV如何调节凋亡机制，从而不仅促进病毒的生命周期，而且可能促进病毒的转化。 相关性：人乳头瘤病毒是宫颈癌的致病因素。了解病毒癌蛋白如何有助于调节病毒生命周期，最终将有助于深入了解HPV是如何导致疾病的，并提供潜在的治疗靶点。

DESCRIPTION (provided by applicant): My career goal is to become a leading independent scientist in the DNA tumor virus field. During the mentored phase of the Pathway to Independence award, I will work to further establish my publication record in the HPV field which will lay the groundwork for the research I will do as a faculty member. This project will provide training with new techniques (confocal microscopy, flow cytometry, gel shifts) that will be applicable to the research I perform as an independent investigator. The expertise of Dr. Laimins, as well as other faculty members in the Microbiology-Immunology Department, will be invaluable in learning the necessary skills to become a successful independent researcher. Human papillomaviruses are small, DNA viruses that are considered the etiological agents of cervical cancer. The infectious life cycle of HPV is dependent on cellular factors and epithelial differentiation. Differentiation triggers the productive phase of the life cycle, which includes viral genome amplification and virion production. The long-term goal of this project is to understand the mechanisms that regulate the activation of differentiation-dependent viral events. I recently demonstrated that HPV-31 stimulates a low level of caspase activation upon differentiation that is characteristic of the mitochondrial apoptotic pathway and necessary for amplification of viral genomes through cleavage of the viral replication protein E1. Based on the finding that caspase activation could be stimulated by both E6 and E7, and the identification of caspase cleavage sites in several HPV proteins, I hypothesize that: HPV, through E6 and E7, induces caspase activation in differentiating cells to modify viral protein function as a mechanism to activate late viral events. In specific Aim 1, to be initiated during the mentored phase, I will identify the mechanism(s) by which HPV proteins activates caspases in differentiating cells by examining the effect of E6 and E7 on mitochondrial membrane integrity and identifying domains in E6 and E7 that are necessary for caspase activation. In Specific Aim 2, to be initiated during the independent phase, I will determine how caspase cleavage of HPV proteins contributes to differentiation-dependent events in the life cycle by using in vitro and in vivo studies. Delineating the pathway by which E6 and E7 mediate caspase activation in differentiating epithelia to result in cleavage of viral proteins will offer insight as to how HPV modulates apoptotic machinery to facilitate not only the viral life cycle, but possibly transformation as well. RELEVANCE: Human papillomaviruses are the causative agents of cervical cancer. Understanding how the viral oncoproteins contribute to regulation of the viral life cycle will ultimately offer insight into how HPV causes disease and provide potential therapeutic targets.