HUMAN PAPILLOMAVIRUS GENE EXPRESSION

人乳头瘤病毒（hpv）引起最普遍的性传播疾病的病毒病因。感染是亚临床或表现为良性乳头状瘤和尖锐湿疣。高风险hpv感染，特别是18型和16型，可能发展为发育不良和癌症。由于HPV仅在处于终末分化的人鳞状上皮细胞中繁殖，我们和其他人采用了培养原代人角质形成细胞（PHKs）的器官型（筏）培养技术来研究HPV基因在体外的功能。我们已经证明HPV-18 E7基因由HPV-18增强子- e6启动子（URR）表达是分化依赖的，并在筏培养的分化phk中重新激活宿主DNA复制。这些结果表明，E7蛋白的功能是促进病毒DNA复制，E7蛋白与pRB结合并使其失活，从而释放E2F:DP转录因子。我们还发现，在分化的phk中，ERR-E7同时诱导细胞周期蛋白E和普遍的细胞周期蛋白依赖性激酶抑制蛋白p21cip1。p21cip1的诱导由转录后机制介导。在筏培养和良性乳头瘤中分化的phk中，这两种宿主蛋白的诱导以及宿主和病毒DNA的合成以一种互斥的方式发生，这解释了患者标本中病毒活性的异质性。此外，某些E7突变激活了具有代表性的e2f应答宿主复制基因，即分化phk中DNA聚合酶α的p180亚基，但不能诱导PCNA （DNA聚合酶δ辅助因子），因此不能诱导细胞DNA合成。这些结果表明，从pRB中释放E2F:DP因子是必要的，但不足以激活所有DNA复制基因。该应用程序旨在深入研究分化角化细胞的机制：(1)E7通过使用一组E7突变激活宿主DNA复制，如pol- α和PCNA，并最终激活宿主DNA复制；(2) E7激活PCNA基因，特别注意第一个内含子的顺式元件、跨越RNA起始位点的YY1结合位点以及启动子区域的其他调控元件的可能作用；(3) E7诱导细胞周期蛋白E和p21cip1蛋白，重点研究细胞周期蛋白E或p21cip1蛋白是否抑制非预定的细胞DNA合成。我们将确定E7的生化特性，如与肿瘤抑制因子pRB、P107、转录因子TBP和YY1的结合，以及酪蛋白激酶II的磷酸化，并将其与刚才描述的生物学后果相关联。这些研究将阐明控制细胞DNA复制机制以及HPV繁殖的途径。

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.