Interaction of simian virus 40 large T-antigen with cellular DNA polymerase alpha: studies with various T-antigen mutants of SV40.
Simian virus 40 (SV40) large T antigen binds to host cell DNA polymerase alpha and the T-antigen-DNA polymerase alpha complex is implicated in the initiation of viral DNA replication. We have examined various SV40 T-antigen mutants to test the correspondence between viral DNA replication and T-antigen-DNA polymerase alpha complex formation. The various SV40 T-antigen mutants were used to either infect or transfect African green monkey kidney cell line CV-1, and at different time intervals we measured the production of T-antigen and host cell DNA polymerase alpha by radioimmunoassay, complex formation by a sandwich radioimmunoassay and the amount of viral DNA synthesis by dot-blot hybridization analysis. There was a good correlation between complex formation and viral DNA synthesis in lytic mutants of SV40. Poor complex formation and correspondingly lower DNA synthesis were observed in the non-viable mutants of SV40, even though significant amounts of T-antigen and DNA polymerase alpha were present. Our results substantiate the earlier findings of T-antigen-DNA polymerase alpha complex formation and establish the need for formation of this complex in promoting viral DNA synthesis.
Pistillo
J M
JM
Department of Biochemistry, University of Nebraska Medical Center, Omaha.
Vishwanatha
J K
JK
D000818:Animals; D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D009154:Mutation; D011485:Protein Binding; D011863:Radioimmunoassay; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004257:DNA Polymerase II