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. 2002 Sep;76(18):9194-206.
doi: 10.1128/jvi.76.18.9194-9206.2002.

Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery

Josephine N Harada  1 , Anna Shevchenko, Andrej Shevchenko, David C Pallas, Arnold J Berk
Affiliations

Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery

Josephine N Harada et al. J Virol. 2002 Sep.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Identification and characterization of novel E1B-55K-associated proteins. (A) HeLa suspension cultures were either mock infected or infected with the Ad5 or dl1520 viruses at an MOI of 100. At 14 h postinfection, nuclear extracts were prepared and immunoprecipitated with the 2A6 anti-E1B-55K antibody (111). Immunoprecipitates were resolved by SDS-PAGE, and the E1B-55K coprecipitating proteins were visualized by staining with silver nitrate. Asterisks are used to indicate where E1B-55K aggregates and breakdown products were identified. The sizes of the molecular mass markers used are indicated at left. (B and C) Nuclear extracts prepared from mock-, Ad5-, or dl1520-infected cells were immunoprecipitated with antibodies directed against the E1B-55K (B and C) or Ad 72K (B) DNA-binding proteins. Immunoprecipitates were resolved by SDS-PAGE, and upon transfer to a nitrocellulose support, Western analyses for the indicated proteins were performed using the appropriate antibodies.
FIG. 2.
FIG. 2.
Gel filtration chromatographic analysis of E1B-55K-containing complexes. (A and B) Nuclear extract prepared from Ad5-infected HeLa cells at 14 h postinfection was resolved on a Superose 6 column. The isocratically eluted fractions were then immunoprecipitated with the 2A6 anti-E1B-55K monoclonal antibody (111). Immunoprecipitates were resolved by SDS-PAGE and either transferred to a nitrocellulose membrane for Western blot analysis (A) or stained with silver nitrate (B). Western blot analyses were performed using the indicated antibodies (A). (A and B) Fraction numbers are listed at top, with the elution positions of the molecular mass standards thyroglobulin (669 kDa), ferritin (440 kDa) and aldolase (158 kDa) denoted by arrows above the fraction numbers. (B) Fraction 6 is duplicated at left. The major proteins bands coeluting with E1B-55K in the 800- to 900-kDa range are indicated at far left, and SDS-PAGE molecular mass markers are indicated at right.
FIG. 3.
FIG. 3.
An E1B-55K-containing complex ubiquitinates p53 in vitro. (A) Nuclear extracts were prepared at 14 h postinfection and immunoprecipitated with the 2A6 anti-E1B-55K antibody. In vitro ubiquitination reactions were prepared by combining the immunoprecipitates with in vitro-transcribed and -translated radiolabeled p53 and an excess of unprogrammed rabbit reticulocyte lysate, as described in Materials and Methods. The reactions were incubated for 4 h at 25°C and then analyzed by SDS-PAGE and autoradiography. Duplicate reactions are shown. Reactions in the right two lanes contained protein A-Sepharose beads without bound antibody that were incubated with nuclear extract from mock-infected cells. Where appropriate, the indicated amounts of purified GST-conjugated ubiquitin (B) and ubiquitin K48R (C) were also added to the reactions. Conjugates of p53 with the various ubiquitin derivatives are denoted by the brackets at the right of each panel, and molecular mass markers are indicated at left.
FIG. 4.
FIG. 4.
E4-orf6 induces the assembly of E1B-55K with Cullin-5, Rbx1/Roc1/Hrt1, and the Elongins B and C. (A) 293 cells were either mock infected or infected with wild-type Ad5 or dl1520 at an MOI of 100. At 14 h postinfection, nuclear extracts were prepared and immunoprecipitated with either the 2A6 anti-E1B-55K hybridoma supernatant (111) or (M186)B6 anti-72K DBP monoclonal antibody (102). The immunoprecipitates were subsequently resolved by SDS-PAGE and Western blotted for the indicated proteins. (B) 293 cells were either mock transfected or transfected with pcDNA3-E4orf6-Flu (85). Nuclear extracts prepared at 42 h posttransfection were immunoprecipitated with the 2A6 anti-E1B-55K hybridoma supernatant (111). Immunoprecipitates were resolved by SDS-PAGE and Western blot analyses performed with the indicated antibodies. (C) Nuclear extract from 293 cells was fractionated on Superose 6, and E1B-55K-containing complexes were immunopurified using the 2A6 anti-E1B-55K antibody (111) as described in the legend to Fig. 2. E1B-55K was detected by Western blotting with the 2A6 hybridoma supernatant (111). Superose 6 fraction numbers are indicated at top. The void volume is denoted with an asterisk, and the elution positions of the molecular mass standards thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa) are indicated by the arrows.
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