The oncogenic papilloma viruses replicate as nuclear plasmids in transformed cells and as such independent replicators, they provide an ideal system to study the mechanisms of DNA replication, chromosome segregation, and gene expression. Understanding the coordination of these processes in the cell cycle is indeed a central goal of molecular biology. We propose to continue to study the ways in which the viral encoded E1 and E2 proteins work to achieve these ends for papilloma viruses. In particular, E2 as the central regulatory protein in the viruses life cycle is known to control DNA replication and gene expression. E2 targets the viral helicase E1 to the ori site and in separate activities interacts with cellular factors to stimulate transcription at many viral promoters. We have recently uncovered a new function of E2 that is regulated by cellular kinases. E2 attaches to mitotic chromosomes and in so doing stabilizes and assures viral genome segregation and nuclear retention in dividing cells. Our specific goals related to these themes are as follows: a) utilize x-ray crystal structure models of the activation domain of E2 (from HPV18) to test notions for how the enhancer works as a transcription factor and DNA replication factor. We are in particular interested in resolving if one region of the molecule can interact simultaneously with different proteins or if the domain of interactions overlap. To do this we will ask which surface residues are actually involved in function. b) We propose to use cell based assays both in drosophila and mammalian cells to explore E2 function and to better define the HPV18 promoter map. c) We want to clarify the physical role of E1 in tethering plasmids (hitch-hiking) to chromosomes; to ask if viral DNA is associated with cellular chromosomes continuously in the cell cycle or preferentially in mitosis. Moreover, we want to have a better understanding of how and when E1 and E2 occupy DNA sites on viral plasmids throughout the cell cycle. d) We want to understand how DNA strands pass through the E1 helicase rings and to further probe the E1 and E2 interaction surfaces. These studies in all will help define regions of the protein that might be targets for drug therapies in patients presenting a high viral load and therefore at risk for cervical cancer.
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