Binding sites for monoclonal antibodies and for mRNPs on SV40 large T‐antigen determined with a cleavage map

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Abstract
Immune complexes of SV 40 large T-antigen with monoclonal papova virus 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 kilodalton 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 messenger ribonucleoprotein (mRNP) 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. 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 mRNP whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.