Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map.
Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity; D007120:Immunochemistry; D010446:Peptide Fragments / Q000276:immunology; D012333:RNA, Messenger / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology*; D013539:Simian virus 40 / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D010446:Peptide Fragments; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D014764:Viral Proteins