Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery.
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.
Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1570, USA.
Josephine N
JN
Harada
Anna
A
Shevchenko
Andrej
A
Shevchenko
David C
DC
Pallas
Arnold J
AJ
Berk
D017501:Adenovirus E1B Proteins / Q000378:metabolism*; D017504:Adenovirus E4 Proteins / Q000378:metabolism; D000260:Adenoviruses, Human / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017501:Adenovirus E1B Proteins; D017504:Adenovirus E4 Proteins; D009097:Multienzyme Complexes; D020543:Proteome; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D014764:Viral Proteins; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases