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.
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