): We have shown that acute expression of the BPV E2 protein causes dramatic repression of HPV18 E6/7 expression and substantial growth inhibition in HPV DNA-positive cervical carcinoma cell lines. The E2 protein mobilizes a complex growth inhibitory program in the cells that includes induction of cdk inhibitors and repression of the cdk activators, cdc25A and B. Genetic studies indicate that the ability of the E2 protein to inhibit proliferation and repress E6/7 an cdc25A/B expression requires an intact transactivation domain. We propose to continue to characterize the effect of the BPV E2 protein on cervical carcinoma cells and to provide a mechanistic explanation for these effects. Many of the experiments will exploit the SV40 recombinant virus system we have described to introduce growth regulatory genes into human cervical carcinoma cells. We will carry out gene transfer, genetic, and biochemical experiments to test the hypothesis that repression of cdc25A expression is required for growth inhibition by the BPV E2 protein. Similar experiments will be carried out to determine whether repression of the expression of HPV E6/E7 and E2F family members is required for growth inhibition by the BPV E2 protein and to clarify the intracellular the intracellular pathways involved in cell cycle control. Particular emphasis will be placed on testing whether all of the growth regulatory effects of the E2 protein are mediated by E6/E7 repression, and on using this system to explore the independent contributions of the E6 and E7 proteins to maintenance of the transformed state. We will also characterize systematically the effects of HPV16, HPV18, HPV31b E2 proteins on cellular physiology to determine whether qualitative differences exist between the E2 proteins isolated from different papillomavirus types. Experiments will be performed to provide a molecular explanation for any differences that exist and to identify physiologically relevant E2 targets in cells. Finally, a variety of experiments will be carried out to determine the mechanism of E2-mediated repression of the cdc25A promoter. These experiments should provide new insights into cell cycle regulation, cervical carcinogenesis and papillomavirus.
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