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Related Experiment Videos

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

C S Sullivan1, S P Gilbert, J M Pipas

  • 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

Journal of Virology
|February 13, 2001
PubMed
Summary

Simian virus 40 large T antigen directly binds heat shock cognate 70 kDa protein (Hsc70) with a 1:1 ratio, requiring Hsc70

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Area of Science:

  • Molecular Biology
  • Virology
  • Oncology

Background:

  • Simian virus 40 large T antigen (SV40 T-ag) is a key oncoprotein driving viral replication and cellular transformation.
  • SV40 T-ag possesses a J domain crucial for its functions, interacting with heat shock cognate 70 kDa protein (Hsc70), a cellular chaperone.
  • The precise nature of the SV40 T-ag and Hsc70 interaction, whether direct or indirect, remained unclear.

Purpose of the Study:

  • To elucidate the direct binding interaction between SV40 T-ag and Hsc70.
  • To characterize the binding stoichiometry, affinity, and nucleotide dependency of the SV40 T-ag–Hsc70 complex.
  • To identify domains within SV40 T-ag and Hsc70 critical for their association.

Main Methods:

  • Direct binding assays to determine stoichiometry and dissociation constants.
  • ATP hydrolysis assays to assess nucleotide dependency.
  • Analysis of T-ag fragments and yeast Hsc70 homologues to map interaction domains.

Main Results:

  • SV40 T-ag directly binds Hsc70 with a 1:1 stoichiometry (Kd = 310 nM Hsc70).
  • Complex formation is ATP hydrolysis-dependent at Hsc70's active site (Kd = 0.16 microM ATP) but independent of T-ag's ATP binding site.
  • A J-domain fragment of T-ag formed a transient, not stable, complex; stable association requires multiple domains from both proteins.

Conclusions:

  • The SV40 T-ag–Hsc70 interaction is specific and requires contributions from multiple domains of both proteins.
  • This study provides the first evidence of nucleotide requirement for SV40 T-ag and Hsc70 association.
  • Findings establish a foundation for studying chaperone-dependent tumorigenesis mediated by SV40 T-ag.

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