Human papillomavirus type 16 (HPV-16) is the most prevalent high risk virus associated with cervical and penile intraepithelial neoplasia and cancers, and HPV-11 is a common low risk genotype primarily causing benign anogenital condylomata and recurrent respiratory (laryngeal) papillomatosis. We propose to use infected keratinocyte culture systems developed in our laboratories to identify and evaluate specific pharmacological drugs that have differential effects on the mRNA transcription, episomal DNA replication, protein function and virion assembly of both virus types. These in vitro systems are also ideal for assessing agents that modulate epithelial proliferation and differentiation and thereby may affect viral activity indirectly. The basic strategy is to grow keratinocytes that contain episomal HPV DNA or are infected with HPV virus on dermal equivalents consisting of collagen matrices and fibroblasts. When the culture is grown at the medium:air interface mimicking the environment of normal skin, the keratinocytes stratify and differentiate. In such organotypic cultures, the complete HPV productive program is recapitulated, exactly as observed in vivo with patient lesions. The cell line W12, which was isolated in the lab of Co-PI MAS and contains episomal HPV-16, will be used as one the test systems. A second system uses primary human cervical and foreskin keratinocytes (PHK) infected with HPV-16 viruses generated from the W12 cells. The third system involves PHK transfected with HPV-11 DNA with high efficiency procedures developed in labs of the PI TRB and Co-PI LTC. Agents effective in modulating epithelial differentiation or viral activities will then be tested directly on raft cultures developed from fresh patient biopsies known to contain HPV DNAs or in grafts in nude mice. Potential modifiers to be tested include cytokines, nucleoside analogs, topoisomerase poisons, antisense or triplex forming oligonucleotides, and suramin, a potent multi-target drug which inhibits the binding of growth factors to cell surface receptors and interferes with host protein kinase C, DNA polymerases and topoisomerase, among others. Additional reagents that are identified by the Antivirals Program Director will also be tested. The assays include histology to monitor epithelial differentiation and to assess possible cellular toxicity, viral pathogenesis, in situ hybridization to measure viral DNA replication and the synthesis of early and late region viral mRNAs as well as host messenger RNAs for which we have constructed specific probes, immunocytochemistry to detect the synthesis of viral and host proteins, and transmission electron microscopy to visualize virions. Additional refinements to the procedures for tissue culturing and DNA transfection into keratinocytes and new probes will be developed as part of this project. In the future, transcription and replication assays in transiently transfected cells or cell-free systems can be used to complement the assays performed on raft cultures when appropriate. The combined results should elucidate the mechanisms of action of promising pharmacological modifiers of viral infection.
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