The early proteins of the small oncogenic DNA viruses have been model viral oncogenes and are studied by both biochemical and genetic approaches. We propose to search for cellular components which interact with these viral proteins. We will focus upon the molecular mechanisms which regulate SV40 and BPV-1 transcription and replication. The use of in vitro biochemical systems should complement our previous genetic approaches and aid in understanding nuclear regulatory events. Some specific projects are listed below. 1. The majority of the highly repeated B2 genes are repressed in rodent cells grown in culture. SV40 T antigen unmasks a number of these genes and leads to the formation of stable transcription complexes. We propose to continue the study of these processes and in particular test models concerning this induction utilizing cell-free systems. 2. We will use an in vitro SV40 replication system to find cellular proteins which complement defective extracts from cells which have lost their abilities to replicate SV40 in vivo. 3. Electroporation techniques will be used to introduce cellular DNA into revertant lines which contain wild-type alleles of T antigen. If the revertant genomes harbor recessive mutations this approach could lead to the cloning of genes interacting with T antigen. 4. The BPV-1 origin of replication is negatively regulated by one of its gene products. We propose to initiate in vitro replication experiments to examine this regulation in detail and to ask if transcription factors are directly involved in BPV-1 replication, as is indicated by in vivo genetic studies. 5. The BPV-1 E2 protein is a site-specific DNA binding protein and induces an enhancer activity in the cis-acting regulatory region of the virus. We propose experiments to test the notion that this DNA folds into a compact preinitiation complex mediated in part by the direct action of the E2 protein.
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