Human papillomaviruses (HPV) cause benign and malignant hyperproliferative disease. A woman infected with HPV 16 (a high risk HPV) has a 200-fold increased risk for the development of cervical cancer while one infected with HPV 6 (a low risk HPV) has a 10-fold increased risk. Notably, HPV 6 is the causative agent of the most prevalent viral sexually transmitted disease, benign hyperproliferative genital warts. Over a million new cases of this disease are diagnosed annually and almost four billion dollars in US health care costs are spent annually. HPVs replicate in the differentiated compartment of the epithelium. The viruses must, therefore, have the ability to either abrogate growth arrest that occurs when the infected cells differentiate or induce differentiated cells to re-enter the cell cycle. While an examination of the high risk viruses has been emphasized, it is imperative that we understand the mechanism of pathogenicity of the low risk viruses, clinically significant pathogens in their own right. Therefore, our focus is to define the interactions between HPV 6 and a differentiating epithelium that dictate the outcome of infection. Because of the interaction of E7 with pivotal cell cycle control proteins, E7 is likely to play a critical role in altering the intracellular environment in such a way as to promote the virus life cycle. For this reason, we have chosen to concentrate on this gene product. In Specific Aim 1 of the proposal we will establish the role of E7 in the HPV 6 life cycle. We will use the intact HPV 6 genome, with a translation termination linker in the E7 gene, to determine the necessity of 6E7 for a) episomal maintenance in primary undifferentiated keratinocytes during the non-productive stage of the life cycle, and b) the HPV-mediated aberrant cellular DNA synthesis in the suprabasal cell layer of organotypic raft cultures during the productive stage of the life cycle. We will also use the raft culture to determine the sufficiency of 6E7 in altering epithelial cell growth. In Specific Aim 2 we will conduct a structure/function analysis of the 6E7 protein to identify regions of E7 pertinent to the virus life cycle. We will analyze the mutants in the context of the intact genome for their necessity in episomal maintenance in undifferentiated cells and for reprogramming suprabasal cells to enter S phase. In Specific Aim 3 we will determine the cellular targets of 6E7 relevant to the life cycle of the virus, with emphasis on the pRb family:E2F-mediated pathways. The effect of 6E7, in the context of the entire genome, on cellular gene expression during the non-productive and productive stages of the life cycle, will be determined. By conducting these experiments under physiological conditions that mimic a natural infection, we expect to make novel contributions on the activities of this important protein.