The long-range goal of this project is to attain a better understanding of the functions of the early gene products of SV40 within the host cell. Of particular concern, is the elucidation of the role viral and host proteins in SV40-induced cellular transformation. Three complementary approaches are proposed to achieve this goal: 1) Point mutations will be systematically generated throughout the early region of SV40 and used to better define the functional domains of the early gene products and their role in transformation. For this purpose, portions of the early region of SV40 DNA cloned in M13 will be subjected to mutagenesis, the sites of specific mutations determined by dideoxy sequencing and selected mutants tested for biological activity. 2) The function of large T antigen in transformed cells will be investigated by examining the interaction of this protein with host replicons. For this purpose, tsA transformed cells will be subjected to a novel procedure of instantaneous synchronization so that the temporal order of cell DNA synthesis at the permissive temperature can be compared to that occurring at the restrictive temperature. The order of replication of total cellular DNA sequences, simple sequence DNA, integrated SV40 DNA, and specific host genes will be analyzed. 3) The role or T antigens in transformation will be investigated by isolating and characterizing host proteins which interact with T antigen(s) to produce the transformed phenotype. For this purpose, T antigen+ revertants, which appear to have lost the transformed phenotype because of alterations of a host function which interacts with T antigen, will be studied. Attempts will be made to correct such defects by fusion of T+ revertants to normal cells, to divide T+ revertants into complementation groups, to isolate host genes encoding transformation factors, and to characterize such genes and their products.
