In vitro DNA synthesis of the small oncogenic DNA viruses provide good model systems for the dissection of cellular proteins involved in replication. Soluble extracts from either human derived HeLa cells or mouse derived FM3A cells can provide all the necessary cellular proteins required for accurate and complete de novo DNA synthesis of bovine papilloma virus. These extracts must be supplemented with the viral encoded E1, which is a helicase, and the viral enhancer E2, an ATP regenerating system, deoxy and ribonucleotides. We intend to fractionate the HeLa cell extracts using conventional biochemical and immunological procedures to ascertain which cellular proteins are necessary and sufficient for BPV-1 replication in vitro. New cellular activities will be purified to homogeneity, and the proteins identified. Peptide sequence data will be generated for these proteins, to aid in gene cloning. In order to fractionate the cellular extracts, we will scale up our procedures to routinely prepare them from 20-50 liters of cell culture material. With this amount of material, fundamental questions related to papilloma virus replication can be addressed. In particular, we are interested to know if the transcription factor E2 interacts both with the cellular replication and transcription machinery, and if a common protein component links the two processes. At this point, we know that the cellular DNA polymerase alpha:primase and/or associated proteins are required in vitro, as are cellular topoisomerases. By analogy to SV-40, which provides the only other eukaryote in vitro replication system that utilizes cellular DNA polymerases, we suspect that many other components are required. These include delta polymerase and associated PCNA, RP-A which is a three subunit single stranded DNA binding protein, RP-C which is a primer binding ATPase, and activating kinases and phosphatases. Preliminary results indicate that other cellular activities not known from the SV-40 system may be important in papilloma replication. Therefore, we will positively identify by immunological criteria those cellular proteins now suspected to be important for BPV-1 replication, purify them in quantity, and add back to them in mixture fractions from replication competent extracts.
Other specific aims i nclude an analysis of the lag phase in the in vitro system, studies on the nature of E1 mediated repression of transcription, and a mapping of the DNA binding domain of E1. Studies addressing the human papilloma virus 6B replication are also described, and the outcome of these experiments will determine future goals.
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