The analysis of human papillomavirus type 6 (HPV-6) in squamous papilloma of the larynx and respiratory tract will be continued to investigate the molecular biology and mechanism of cellular transformation of these viruses. Analysis of the viral DNA in biopsy specimens has shown an association of one subtype with a more severe disease. These studies will be extended to a larger population of patients to clarify the role of viral subtypes in establishing disease. The structural characterization of the four subtypes of HPV-6 genomes that have been molecularly cloned from laryngeal papillomata will be pursued by comparing the DNA sequence of diverged regions identified among the subtypes. Examination of DNA sequences will reveal if base substitutions alter predicted amino acid sequences, open reading frames, or potential splice sites that would alter protein products and account for behavioral differences among these viruses. To isolate and characterize HPV-6 transcripts that have been identified by Northern analysis and to detect low abundance mRNAs, cDNA libraries will be constructed from RNA isolated from respiratory tract lesions. The size, structure and location on the viral genome of the cDNA inserts will be determined by fine structure mapping and hybridization analyses. Characterized cDNA clones will be inserted into appropriate vectors for expression in prokaryotic and eukaryotic cells to produce the protein products of HPV-6 genomes, since understanding the mechanism of transformation requires the identification of the protein(s) involved. Monoclonal antibodies will be produced to nuclear proteins of laryngeal papillomata to develop immunological probes to identify viral capsid and non-structural proteins. These antibodies will also be used to analyze proteins produced by recombinants in the cDNA expression library to establish structural/functional maps of the HPV-6 genome. Constructs will be made with dominant selectable markers and transfected into human primary epithelial cells and other recipient cells to establish an in vitro model system to investigate the oncogenic potential and biological activity of HPV-6 DNA. Selected cells will be assayed for expression of viral sequences and parameters of transformation to investigate the virus-cell interactions that are important in the replication of these human tumor viruses.
