TRANSCRIPTIONAL REGULATION DURING THE PAPILLOMAVIRUS LIFE CYCLE AND ELIMINATION OF INFECTION USING HOMOLOGOUS RECOMBINATION

Human Papillomaviruses (HPVs) require terminal differentiation of the host cell to produce infectious virions. The process of viral maturation involves a variety of changes in the expression/activity of host proteins that lead to high-level replication of the viral genome and expression of the late viral genes. Although the late promoter regions of HPV-16 are still not fully characterized, differentiation-dependent regulation of viral genes is thought to involve changes in the binding of host cell transcription factors to the viral promoter and regulatory regions. Currently, little is known about specific cellular transcription factors involved in this process. We have used the Panomics TransSignal Protein/DNA array to identify changes in the levels of cellular transcription factors during methylcellulose-induced differentiation of W12 (20863) cells containing HPV-16. We then identified the differentially expressed transcription factors that specifically bind to HPV-16 promoters. We have validated the results obtained from the Panomics array by Western blot analysis and with chromatin immunoprecipitation. This approach identified approximately thirty transcription factors, many of which represent novel viral DNA-host protein interactions. At present, no treatments exist that effectively target and eliminate papillomaviruses (PVs) from infected cells or prevent its replication. We are employing a strategy to prevent virus replication in PV-infected cells through the conditional expression of the herpes simplex virus type 1 thymidine kinase (TK) gene. Expression of TK in this system is expected to be triggered by a homologous recombination event between the endogenous PV genome and a nonexpressing TK gene cassette, which is expected to change the nonexpressing cassette into a form that expresses TK. Various constructs were generated to express the TK in the above manner. Transfection of cell lines with a TK nonexpressing plasmid did not result in TK production. However, cotransfection of cell lines with PV plasmids along with the above TK construct containing PV sequences resulted in TK expression as shown by Northern and Western blot analyses. We also developed a TK expression cassette utilizing an adeno-associated virus (AAV) vector. Delivery of the cassette by AAV to PV-infected cells resulted in TK expression, and ganciclovir treatment resulted in efficient killing of these cells