The long term goal is to investigate the mechanisms that regulate early and late viral gene expression of oncogenic human papillomaviruses. Papillomaviruses are small DNA viruses which induce a variety of proliferative epithelial lesions. Over seventy different types have been identified and a subset of these, including types 16, 18, and 31, are the etiologic agents of cervical cancers. Papillomaviruses infect the basal cells of the epithelia and establish their genomes as low copy episomes. The productive life cycle of these viruses is linked to epithelial differentiation with the induction of late gene expression, genome amplification, and assembly of virions restricted to suprabasal cells. The use of organotypic raft culture has allowed for the study of the differentiation-dependent aspects of the viral life cycle. Several years ago, the applicant used raft cultures to synthesize human papillomaviruses from a continuous cell line that had been derived from a patient biopsy. Recently, he developed methods to synthesize oncogenic human papillomaviruses from transfected cloned DNAs. These methods now allow him to perform a detailed genetic analysis of the productive life cycle of human papillomaviruses and will be used extensively in the proposed studies. He has used these methods for genetic analysis to demonstrate a requirement for episomal templates in the induction of high levels of late gene expression. In addition, a set of mutant HPV 31 E2 proteins were identified which retain replication ability but have lost transcriptional activation function. HPV 31 genomes containing these mutations are able to induce late genes in raft cultures but exhibited a reduced copy number. Finally, he has developed methods for the rapid differentiation of HPV positive cells in methylcellulose to induce differentiation-depended late functions. These new methods and observations form the basis of the proposed studies on the regulation of HPV gene expression. The applicant will ask the following questions: I. What are the targets of HPV 31 E2 transactivation function during the productive viral life cycle? II. What signals control differentiation-dependent late expression from the p742 promoter? III. What mechanisms regulate early and late HPV polyadenylation?
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