Immunoprecipitation of the simian virus 40 late transcription complex with antibody against T-antigen.
Tumor antigen (T-antigen) of simian virus 40 (SV40) has been shown to have a number of regulatory roles in both viral replication and early viral transcription. However, the nature of its role on late viral transcription remains unclear. We have analyzed for the presence of T-antigen on SV40 late viral transcription complexes which exhibit RNA polymerase II extension activity in vitro. Nuclear extract or glycerol gradient-isolated transcription complexes were treated with either polyclonal or monoclonal antibodies, and the amount of extension activity that could be immunoprecipitated was determined. Anti-T antibody derived from hamster ascites as well as the anti-T monoclonal antibodies PAb 102, 109, 416, and 419 all precipitated 12-29% of viral transcription complex activity. Immunoprecipitation resulted in significant enrichment of transcription complex activity relative to bulk minichromosomes, indicating a preferential association of T-antigen with the late viral transcription complex. This is the first direct demonstration of the presence of T-antigen on the SV40 late transcription complex. Furthermore, the immunoprecipitated transcription complexes exhibited a salt dependence of their in vitro extension activity which was distinct from that of the total complex population, indicating that T-antigen is present on a specific subclass of transcription complexes.
Hadlock
K G
KG
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor 48109-0010.
Quasney
M W
MW
Lutter
L C
LC
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007163:Immunosorbent Techniques; D009693:Nucleic Acid Hybridization; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D012319:RNA Polymerase II / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D014158:Transcription, Genetic; D014775:Virus Activation
D000906:Antibodies; D000911:Antibodies, Monoclonal; D009856:Oncogene Proteins, Viral; D012367:RNA, Viral; D012319:RNA Polymerase II