CONSTRUCTION OF SV40 DELETION MUTANTS AND DELIMITATION OF THE BINDING DOMAIN FOR HEAT-SHOCK PROTEIN TO THE AMINO-TERMINUS OF LARGE T-ANTIGEN

SV40 large T-antigen (T-ag) mutants were generated using a cassette mutagenesis strategy and naturally occurring restriction sites. T-ag mutant constructs included internal in-frame deletions, frame-shift deletions that resulted in amino-terminal fragments, and internal initiation mutants that produced carboxy-terminal fragments; no foreign amino acids were introduced. The deletion mutants were stably expressed in BALB/c 3T3E cells and were analyzed for ability to bind heat shock cognate protein 70 using an ATP release assay of T-ag immunoprecipitates. Complex formation between heat shock protein and T-ag was independent of p53 involvement. The heat shock protein binding domain was narrowed to the amino-terminal 97 amino acids of T-ag, with the first 29 residues influencing the interaction. The amino-terminal domain of T-ag is important in both viral replication and cell transformation. We propose that the functional interactions of this highly interactive region of T-ag may be modulated by heat shock cognate protein 70.