During the tenure of the previous granting period we, as well as others, have demonstrated the presence of human papillomavirus (HPV) DNA in a variety of premalignant and malignant tumors from patients with wart disease syndromes. Since the association of HPV DNA with several of these malignant tumors is now established, the most important issue becomes whether or not these viruses contribute to the malignant phenotype. Thus, the major objective of this continuing grant application is to determine the involvement, if any, of human papillomaviruses in the progression of certain chronic or recurring wart disease syndromes to the malignant phenotype. To this end our renewal application proposes to: 1) complete our nucleotide sequencing of the HPV DNA species that we have most consistently found associated with malignant tumors both primary and metastatic (i.e., HPV-5 DNA). We also plan to complete nucleotide sequence analysis of the pertinent regions of the subgenomic and recombinant forms of HPV DNA that we have isolated in an effort to determine the possible mechanism(s) by which these variant forms of HPV DNA are derived; 2) continue our comparative analysis of the expression of HPV in both benign and malignant tissues to determine the specific HPV genetic sequences that might be involved in malignant progression; 3) alternatively, we plan to employ genetic dissection and site-specific mutagenesis of papillomavirus DNA to identify directly those nucleotide sequences responsible for malignant progression by testing such genetically altered DNAs in a DNA transfection assay in vivo using an experimental rabbit model system that we have recently developed in our laboratory; 4) molecularly clone to expression the capsid proteins of HPV-5 with the ultimate objective of determining whether such proteins can reduce the incidence of wart disease in high risk patients and concommitantly reduce the rate of malignant progression; and 5) analyze HPV gene expression in normal skin cells to determine the nature of the HPV genetic sequences required for the maintenance of HPV DNA in these cells. The methodologies that we plan to employ to achieve these objectives are for the most part already implemented in our laboratory and include a variety of physiochemical, enzymological and filter hybridization techniques available for the analysis of DNA, RNA and protein including nucleotide sequence analysis.
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