Human papilloma viruses (HPVs) of many genetic types cause hyperproliferations of either cutaneous and mucosal epithelia. Of particular medical concern are those types associated with venereally transmitted genital infections that can lead to benign condylomata, dysplasias, carcinoma-in-situ and metastatic tumors. They cause the cytological changes for which Pap smears screen. The long term goals of the proposed project are to understand the reciprocal interactions of viral and host keratinocyte gene expression and tissue differentiation. The immediate ojectives are to decipher the patterns of HPV transcription and RNA processing, the structures and biochemical functions of the encoded proteins, and the positive and negative regulation of the various promoters for early and late transcripts. The proposed studies are (1) to define the transcriptional enhancer sequences of HPV type 1; (2) to explore the molecular interactions between the HPV-1 and -11 enhancer sequences and positive transactivators, the E2 proteins of HPV-1 and -11, as well as a putative negative factor, the carboxy-terminal half of the HPV-11 E2 protein (E2-C), using proteins generated from expression vectors in E. coli and in mammalian cells; (3) to determine which of the several promoters identified in HPV-1 and -11 warts are responsive to the regulation of the E2 and E2-C proteins; (4) to map the mRNA structures of HPV-16, -18 (and other prevalent types) associated with dysplasias and carcinomas; based on these data, mRNA exon-specific probes will be constructed and the viral genetic expression examined by in situ hybridization of patient biopsies; the transcriptional patterns will be correlated with the disease manifestation; (5) to prepare cDNA clones of transcripts from HPV-16, -18 (and othe types) to determine how the open reading frames (ORFs) are spliced and utilized; (6) to express HPV-16 and -18 early region ORFs and cDNAs in E, coli and prepare antibodies against them; (7) to detect and localize HPV proteins in patient biopsy specimens by Western blots, immunocytochemistry and immunoelectron microscopy; (8) to introduce viral cDNAs and ORFs via recombinant retroviruses into human foreskin for implantation in nude mice (the only in vivo model system for human papilloma viruses) in order to decipher their natural functions; and (9) to follow the infectious cycle of human papilloma virus in human foreskin implanted in the nude mice and the development of condylomata induced.
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