HPV DIFFERENTIATION DEPENDENT REPLICATION

在世界范围内，宫颈癌是女性癌症导致的第二大死亡原因。在所有检查的宫颈癌中，超过90%与人类乳头瘤病毒(HPV)有关。工作假说是，HPV依赖于分化的复制周期的所有方面都受其自然宿主组织鳞状上皮的时空生长机制控制。因此，宿主细胞的分化状态决定了基因表达、病毒DNA复制和病毒粒子形态发生的时空调控。HPV分化依赖生命的两个领域是本应用的重点：第一，描述在病毒生命周期的不同阶段对病毒转录控制至关重要的顺式元件；第二，定义病毒蛋白在终末分化宿主组织中的表达。在第一个特定目标中，HPV突变体将被用来利用体外模型系统来研究转录控制：(I)病毒生命周期的初始阶段；(Ii)宿主上皮组织的分化；(Iii)病毒反式激活蛋白E2的作用；以及(Iv)完整的HPV生命周期。研究HPV基因组中的突变对病毒转录、病毒DNA复制和晚期基因表达的影响是研究这些人类致癌病毒生物学的突破性进展。这些研究是首次提出使用能够繁殖传染性HPV颗粒的体外系统对HPV进行突变分析。在第二个特定目标中，我们将确定HPV蛋白在整个病毒生命周期中分化上皮细胞的时间和空间表达。使用允许用于整个HPV生命周期的器官类型(RAFT)培养系统，包括合成具有感染性的病毒颗粒，将是这些研究的组成部分。RAFT培养系统复制完整的HPV生命周期的能力是一项技术进步，能够为整个HPV生命周期中病毒转录和蛋白表达的调控提供重要和新颖的见解。当拟议的研究完成后，我们预计将确定位于主要HPV31b启动子上游的顺式调控元件在病毒转录控制、病毒DNA复制和病毒粒子合成中的作用。此外，我们希望确定HPV31b蛋白的时间和空间表达模式，并将病毒蛋白表达与转录本表达、启动子使用、上皮分化特异性蛋白的表达以及病毒粒子的形态发生联系起来。

Worldwide, cervical cancer is the second leading cause of death due to cancer in females. Human papillomaviruses (HPV) have been associated with over 90 percent of all cervical cancers examined. The working hypothesis is that all aspects of HPV's differentiation-dependent replication cycle are controlled by the temporal and spatial growth mechanisms of its natural host tissue, squamous epithelium. Therefore, temporal and spatial controls on gene expression, viral DNA replication, and virion morphogenesis are stipulated by the differentiation state of the host cell. Two areas of the HPV differentiation-dependent life that are the focus of this application include: first, describing the cis elements important for viral transcriptional control during different stages of the viral life cycle and second, defining viral protein expression in terminally differentiating host tissue. In the first specific aim, HPV mutants will be utilized to investigate transcriptional control using in vitro model systems for (i) the initial stage of the viral life cycle; (ii) the differentiation of the host epithelial tissue; (iii) the contribution of the viral transactivating protein, E2; and (iv) the complete HPV life cycle. The ability to study mutations incorporated into the HPV genome on viral transcription, viral DNA replication, and late gene expression represents a breakthrough advance for investigating the biology of these human oncogenic viruses. These studies represent the first proposed mutational analysis of a HPV using a permissive in vitro system that is capable of propagating infectious HPV particles. In the second specific aim, we will define the temporal and spatial expression of HPV proteins in differentiating epithelium during the complete viral life cycle. The use of the organotypic (raft) culture system permissive for the complete HPV life cycle, including the synthesis of infectious viral particles, will be an integral part of these studies. The ability of the raft culture system to reproduce the complete HPV life cycle is a technical advance capable of providing important and novel insights concerning the regulation of viral transcription and protein expression during the complete HPV life cycle. When the proposed studies are completed we expect to have defined the role of cis regulatory elements located upstream of the major HPV31b promoter in viral transcriptional control, viral DNA replication, and virion synthesis. Additionally, we expect to have defined the temporal and spatial expression patterns of HPV31b proteins and to correlate viral protein expression with transcript expression, promoter usage, expression of epithelial differentiation-specific proteins, and virion morphogenesis.