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

SV40 large T-antigen (T-ag) mutants were generated using a cassette mu tagenesis strategy and naturally occurring restriction sites. T-ag mut ant constructs included internal in-frame deletions, frame-shift delet ions that resulted in amino-terminal fragments, and internal initiatio n mutants that produced carboxy-terminal fragments; no foreign amino a cids were introduced. The deletion mutants were stably expressed in BA LB/c 3T3E cells and were analyzed for ability to bind heat shock cogna te protein 70 using an ATP release assay of T-ag immunoprecipitates. C omplex formation between heat shock protein and T-ag was independent o f p53 involvement. The heat shock protein binding domain was narrowed to the amino-terminal 97 amino acids of T-ag, with the first 29 residu es influencing the interaction. The amino-terminal domain of T-ag is i mportant 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.