ATP-dependent simian virus 40 T-antigen-Hsc70 complex formation

Simian virus 40 large T antigen is a multifunctional oncoprotein that is re quired for numerous viral functions and the induction of cellular transform ation. T antigen contains a J domain that is required for many of its activ ities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hs c70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (d issociation constant = 310 nM Hsc70). Furthermore, the T-antigen-Hsc70 comp lex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 muM), but T-antigen-Hsc70 complex formati on does not require nucleotide hydrolysis at the T-antigen ATP binding site . N136, a J domain-containing fragment of T antigen, does not stably associ ate with Hsc70 but can farm a transient complex as assayed by centrifugatia n analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing t he ATPase domain. These results provide direct evidence that the T-antigen- Hsc70 interaction is specific and that this association requires multiple d omains of both T antigen and Hsc70. This is the first demonstration of a nu cleotide requirement for the association of T antigen and Hsc70 and lays th e foundation for future reconstitution studies of chaperone-dependent tumor igenesis induced by T antigen.