HPV DIFFERENTIATION DEPENDENT REPLICATION

在世界范围内，宫颈癌是女性癌症死亡的第二大原因。 人乳头瘤病毒（HPV）与90%以上的宫颈癌有关。工作假设是HPV的分化依赖性复制周期的所有方面都受到其天然宿主组织鳞状上皮的时间和空间生长机制的控制。 因此，宿主细胞的分化状态规定了对基因表达、病毒DNA复制和病毒粒子形态发生的时间和空间控制。 作为本申请的焦点的HPV分化依赖性生命的两个领域包括：第一，描述在病毒生命周期的不同阶段期间对病毒转录控制重要的顺式元件，第二，定义终末分化宿主组织中的病毒蛋白表达。 在第一个具体目标中，HPV突变体将用于使用体外模型系统研究转录控制，用于（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的贡献;和（iv）完整的HPV生命周期。 研究HPV基因组中病毒转录、病毒DNA复制和晚期基因表达的突变的能力代表了研究这些人类致癌病毒生物学的突破性进展。 这些研究代表了首次提出的使用能够繁殖感染性HPV颗粒的容许体外系统进行HPV突变分析。 在第二个具体目标中，我们将定义HPV蛋白在完整病毒生命周期中分化上皮中的时间和空间表达。 允许完整HPV生命周期（包括感染性病毒颗粒的合成）的器官型（筏）培养系统的使用将是这些研究的组成部分。 筏培养系统再现完整HPV生命周期的能力是一项技术进步，能够提供关于完整HPV生命周期期间病毒转录和蛋白质表达调控的重要和新颖的见解。 当拟议的研究完成后，我们希望已经确定的作用，顺式调控元件位于上游的主要HPV 31 b启动子在病毒转录控制，病毒DNA复制，和病毒粒子合成。 此外，我们期望已经确定了HPV 31 b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和病毒粒子形态发生相关联。

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.