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.
Sawai
E T
ET
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030.
Rasmussen
G
G
Butel
J S
JS
D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D011485:Protein Binding / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D006360:Heat-Shock Proteins