The goal of the proposed research is to clarify the role of the viral genome in transformation by the small DNA tumor virus, simian virus 40 (SV40). The viral genome and viral gene expression will be studied in phenotypically normal revertants derived from an SV40 transformed mouse cell line, SVT2. The revertant cell lines contain integrated SV40 DNA but exhibit a homeostatic pattern of G1 arrest in confluence culture and are not tumorigenic. The two growth properties distinguish these revertant lines from SVT2 and also from other revertant lines previously derived from SV40 transformed mouse cells. The revertants display the nuclear SV40 large T antigens in a growth-dependent manner; rapidly growing cells have detectable T antigen whereas quiescent cells do not. Hybrid cells producing monoclonal antibodies against SV40 T antigens have been isolated to analyze the virus induced proteins present in the revertant cells. The proposed research seeks to answer a series of specific questions about the viral genome in the revertants. Is the SV40 DNA or its site of integration altered in the revertants? Is there a specific region of the viral genome which is expressed in transformed SVT2 cells but not in the revertants? Is a specific region of the viral genome expressed in the revertants when the cells are growing but not when quiescent? If there is a portion of the SV40 genome which is not expressed, is this controlled at the level of transcription or later? Are any viral gene products altered in amount or physical properties in growing revertant cells? Are any virus-specific proteins altered only in G1 inhibited revertant cells? These questions will be investigated using the techniques of immunoprecipitation, gel electrophoresis, affinity chromatography, restriction endonuclease cleavage, and nucleic acid hybridization.
