HUMAN PAPILLOMAVIRUS GENE EXPRESSION

人乳头瘤病毒（HPV）是最常见的病毒性性传播疾病。感染是亚临床的或表现为良性乳头状瘤和湿疣。感染高危型HPV，特别是18型和16型，可发展为发育不良和癌症。因为HPV仅在经历终末分化的人类鳞状上皮细胞中繁殖，我们和其他人已经采用了原代人类角质形成细胞（PHK）的器官型（筏）培养技术来研究HPV基因的体外功能。我们已经证明，HPV-18增强子-E6启动子（URR）表达的HPV-18 E7基因是分化依赖性的，并在筏培养中重新激活分化的PHK中的宿主DNA复制。这些结果表明，E7蛋白的功能是促进病毒DNA复制，该蛋白结合pRB并使pRB失活以释放E2 F：DP转录因子。我们还发现，ERR-E7同时诱导，在分化的PHK，细胞周期蛋白E和通用的细胞周期蛋白依赖性激酶抑制p21 cip 1蛋白。p21 cip 1的诱导是由转录后机制介导的。诱导这两种宿主蛋白的一方面和主机和病毒DNA合成的另一方面发生在一个相互排斥的方式在分化的PHK在筏文化和良性乳头状瘤，占患者标本中的病毒活动的异质性。此外，某些E7突变激活了代表性的E2 F应答宿主复制基因，即分化的PHK中DNA聚合酶α的p180亚基，但不能诱导PCNA（DNA聚合酶δ辅因子），因此不能诱导细胞DNA合成。这些结果表明，从pRB释放E2 F：DP因子是必要的，但不足以激活所有的DNA复制基因。本申请旨在深入研究在分化的角质形成细胞中的机制，其中：（1）通过使用一组E7突变，E7激活宿主DNA复制，如pol-alpha和PCNA，并最终激活宿主DNA复制;（2）E7激活PCNA基因，特别注意第一内含子中的顺式元件的可能作用，YY 1结合位点跨越RNA起始位点，以及启动子区域中的额外调节元件;和（3）E7诱导细胞周期蛋白E和p21 cip 1蛋白，重点在于非程序细胞DNA合成是否被细胞周期蛋白E或p21 cip 1蛋白抑制。E7的生物化学性质，例如与肿瘤抑制因子pRB、P107和转录因子TBP和YY 1的结合，以及通过酪蛋白激酶II的磷酸化，将被确定并与刚才描述的生物学结果相关联。这些研究将揭示参与控制细胞DNA复制机制以及HPV复制的途径。

Human papillomaviruses (HPVs) cause the most prevalent sexually transmitted diseases of viral etiology. Infections are either subclinical or manifested as benign papillomas and condylomata. Infects by the high-risk HPVs, notably types 18 and 16, cna progress to dysplasia and cancers. Because HPVs propagate only in human squamous epithelia undergoing terminal differentiation, we and others have adapted the technique of growing organotypic (raft) cultures of primary human keratinocytes (PHKs) to investigate HPV gene functions in vitro. We have shown that HPV-18 E7 gene expressed from HPV-18 enhancer-E6 promoter (URR) is differentiation-dependent and reactivates host DNA replication in differentiated PHKs in raft cultures. These results demonstrate that the function of the E7 protein, which binds to and inactivates pRB to release the E2F:DP transcription factors, is to facilitate viral DNA replication. We also found that ERR-E7 simultaneously induces, in differentiated PHKs, cyclin E and the universal cyclin-dependent kinase inhibitory p21cip1 protein. Induction of p21cip1 is mediated by post-transcriptional mechanisms. The induction of these two host proteins on the one hand and host and viral DNA synthesis on the other takes place in a mutually exclusive manner in differentiated PHKs in raft cultures and in benign papillomas, accounting for the heterogeneity of viral activities in patient specimens. Furthermore, certain E7 mutations activate a representative E2F-responsive host replication gene, the p180 subunit of the DNA polymerase alpha in differentiated PHKs but are unable to induce PCNA (a DNA polymerase delta co-factor) and therefore cellular DNA synthesis. These results suggest that the release of E2F:DP factors from pRB is necessary but not sufficient to activate all the DNA replication genes. This application is to investigate in depth the mechanisms in differentiated keratinocytes by which: (1) E7 activates host DNA replication such as pol-alpha and PCNA, and ultimately host DNA replication by using a panel of E7 mutations; (2) E7 activates the PCNA gene, with special attention to possible roles of cis elements in the first intron, the YY1 binding site which spans the RNA initiation sites, as well as additional regulatory elements in the promoter region; and (3) E7 induces cyclin E and p21cip1 proteins, with an emphasis on whether unscheduled cellular DNA synthesis in inhibited by the cyclin E or by the p21cip1 protein. The biochemical properties of E7 such as binding to tumor suppressors pRB, P107, and transcription factors TBP and YY1, and phosphorylation by casein kinase II will be determined and correlated with the biological consequences in just described. These studies will shed light on the pathways involved in controlling the cellular DNA replication machinery as well as HPV reproduction.