We have mapped by S1 and ExoVII digestion and by primer extension viral transcripts in nonvirus producing domestic rabbit papillomas and carcinomas and in virus producing domestic rabbit papillomas and determined their coding capacity. The major transcripts can code for E7 and a truncated E6 protein, and minor ones were identified which can code for E2 and a full sized E6 protein. All early RNA is transcribed from promotors with TATA boxes. Late transcripts of 2.6 and 4.8 kb unique to virus producing tumors code for an L1 and an L2 or possibly an L2-L1 fusion protein. Both share the same leader exon and a capsite located in the untranslated region which is not preceded by a TATA box. This indicates that a switch from early to late transcription involves recognition of a new promotor and suppression of transcription termination at the early polyadenylation site. Expression of early proteins in COS-7 cells has permitted us to identify the E6 protein and preliminary data have also been obtained for E2. DNA analysis of HPV-16 in cervical cancer has shown that in a majority, the HPV-16 DNA is integrated. Some also contain plasmid DNA. Transcript mapping indicated that the major transcripts code for E7 and E6 and it suggests that only integrated DNA is functional and integration, at least of some DNA, may be required for cancer progression. The E7 protein of HPV-16 has been identified as a cytoplasmic phosphoprotein with a short half life. Cottontail rabbit papillomavirus provides the only animal model system and our long term objective is to understand the molecular biology of the virus and particularly how the virus contributes to the development of malignant tumors. In the present application, we propose to: 1) Identify and biochemically characterize ealy viral proteins with respect to subcellular distribution, modification, association with cellular proteins and possible enzymatic activity; 2) Determine their biological function through a genetic analysis in animals and epithelial cell culture; 3) Initiate experiments related to immunologic phenomena.
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