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J Virol, April 1998, p. 3146-3154, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Adenovirus E1B 55K Represses p53 Activation In Vitrodagger

Michelle E. D. Martin and Arnold J. Berk*

Molecular Biology Institute, Department of Microbiology and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90095-1570

Received 12 September 1997/Accepted 6 January 1998

Adenovirus E1B 55K protein cooperates with E1A gene products to induce cell transformation. E1B 55K mediates its effects by binding to and inhibiting the transcriptional activation and growth-suppression functions of the tumor suppressor p53. Previous studies in vivo have suggested that E1B 55K has an active role in repressing p53 transcriptional activation and that this repression function is directed to specific promoters through E1B 55K's interaction with DNA-bound p53. Flag-tagged E1B 55K (e55K) was expressed with the baculovirus expression system and immunopurified. Gel filtration, velocity sedimentation centrifugation, and glutaraldehyde cross-linking indicated that e55K is a dimer with a nonglobular conformation. e55K bound directly to purified p53, causing an ~10-fold increase in p53 affinity for tandem binding sites. Using in vitro transcription assays reconstituted with purified p53, e55K, and HeLa cell nuclear extracts, we found that e55K specifically repressed p53 activation. These results demonstrate that as postulated from earlier transient expression experiments, E1B 55K is a specific repressor of transcription from a promoter with bound p53. Since HeLa nuclear extracts contain little detectable histone protein, E1B 55K probably represses transcription through direct or indirect interactions with the RNA polymerase II transcription machinery.

* Corresponding author. Mailing address: Molecular Biology Institute, 405 Hilgard Ave., UCLA, Los Angeles, CA 90095-1570. Phone: (310) 206-6298. Fax: (310) 206-7286. E-mail: berk{at}ewald.mbi.ucla.edu.

dagger This paper is dedicated to the memory of Carol Newman.




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