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Journal of Virology, September 2002, p. 9194-9206, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9194-9206.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Analysis of the Adenovirus E1B-55K-Anchored Proteome Reveals Its Link to Ubiquitination Machinery

Josephine N. Harada,1 Anna Shevchenko,2 Andrej Shevchenko,2 David C. Pallas,3 and Arnold J. Berk1,4*

Molecular Biology Institute,1 Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California,4 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany,2 Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia3

Received 17 April 2002/ Accepted 12 June 2002

During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-{alpha}1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

* Corresponding author. Mailing address: Molecular Biology Institute, University of California, Los Angeles, 611 Charles Young Dr. East, 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. 9194-9206, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9194-9206.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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