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Journal of Virology, September 2002, p. 9186-9193, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9186-9193.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vivo Association of Adenovirus Large E1A Protein with the Human Mediator Complex in Adenovirus-Infected and -Transformed Cells

Gang Wang and Arnold J. Berk*

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

Received 11 April 2002/ Accepted 11 June 2002

The adenovirus large E1A protein activates transcription from early viral promoters by a mechanism that requires a forty amino acid zinc finger activation domain in E1A conserved region 3 (CR3). Recent results indicate that activation by a Gal4 DNA-binding domain-E1A-CR3 fusion requires an interaction between the E1A-CR3 zinc finger and the Sur2 subunit of the mammalian Mediator (of transcription) complex. Although several host proteins have been shown to bind stably to E1A proteins in adenovirus-infected and -transformed cells, an in vivo interaction with Mediator complex subunits has not been described previously. Using immunoprecipitation and gel filtration analyses of nuclear extracts prepared from HeLa cells infected with adenovirus 5 or mutants that express either large or small E1A specifically and from adenovirus 5-transformed cells, we report here that large E1A, but not small E1A, binds to Mediator complex in vivo. Only ~1 to 10% of large E1A is bound to Mediator complex at 18 h postinfection and in transformed cells, probably explaining why Mediator complex subunits were not identified among cellular E1A-binding proteins described earlier. Surprisingly, even though extracted Mediator can quantitatively bind to an E1A-CR3 affinity column, only on the order of 1% of cellular Mediator complex is bound by E1A in vivo. Much of the large E1A bound to Mediator in 293 cells is in a stable complex that includes RNA polymerase II, leading us to suggest that the interaction of E1A-CR3 with Mediator stabilizes the interaction of Mediator with the polymerase. This stabilization of the interaction between Mediator and RNA polymerase II may contribute to the mechanism of activation by E1A-CR3.

* Corresponding author. Mailing address: University of California, Los Angeles, Molecular Biology Institute, 611 Charles Young Dr., E, Box 951570, Los Angeles, CA 90095-1570. Phone: (310) 206-6298. Fax: (310) 206-7286. E-mail: berk{at}mbi.ucla.edu.

Journal of Virology, September 2002, p. 9186-9193, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9186-9193.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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