Adenovirus L-E1A activates transcription through mediator complex-dependent recruitment of the super elongation complex

Abstract

The adenovirus large E1A (L-E1A) protein is a prototypical transcriptional activator, and it functions through the action of a conserved transcriptional activation domain, CR3. CR3 interacts with a mediator subunit, MED23, that has been linked to the transcriptional activity of CR3. Our unbiased proteomic analysis revealed that human adenovirus 5 (HAdv5) L-E1A was associated with many mediator subunits. In MED23-depleted cells and in Med23 knockout (KO) cells, L-E1A was deficient in association with other mediator subunits, suggesting that MED23 links CR3 with the mediator complex. Short interfering RNA (siRNA)-mediated depletion of several mediator subunits suggested differential effects of various subunits on transcriptional activation of HAdv5 early genes. In addition to MED23, mediator subunits such as MED14 and MED26 were also essential for the transcription of HAdv5 early genes. The L-E1A proteome contained MED26-associated super elongation complex. The catalytic component of the elongation complex, CDK9, was important for the transcriptional activity of L-E1A and HAdv5 replication. Our results suggest that L-E1A-mediated transcriptional activation involves a transcriptional elongation step, like HIV Tat, and constitutes a therapeutic target for inhibition of HAdv replication.

Fig 1

Western blot analysis of mediator subunits associated with L-E1A. (A) Analysis of subunits detected by LC-MS/MS analysis. (B and C) Analysis of subunits that were not detected by LC-MS/MS. Whole-cell extracts or samples immunoprecipitated with the Flag Ab from mock-infected human KB cells or cells infected with HAdv5-12S (Flag/HA-tagged S-E1A, HF-12S) or HAdv5-13S (HF-13S) were probed with Abs specific to the indicated mediator subunits. MED15 was detected by LC-MS/MS analysis but was not analyzed by Western blotting. WCL, whole-cell lysates; CLN C, cyclin C.

Fig 2

Interaction of mediator subunits with L-E1A in MED23-deficient cells. (A) Interaction in MED23-depleted HeLa cells. HeLa cells were transfected with pools of control siRNA or MED23 siRNA, and 36 h after transfection cells were infected with HF-13S (100 PFU/cell). Seventeen h after infection, whole-cell extracts were prepared, immunoprecipitated with the Flag Ab, and analyzed as described in the legend to Fig. 1. (B) Interaction in MEFs. wt MEFs or Med23 KO MEFs were infected with HAdv5-dl312, HF-12S, or HF-13S (500 PFU/cell) and immunoprecipitated with the Flag Ab and analyzed for selected mediator subunits.

Fig 3

Role of mediator subunits in HAdv5 early gene expression. (A and B) Role of Med23. (A) wt MEFs and Med23 KO MEFs were infected with HAdv5-dl312, HF-12S, or HF-13S, and the expression of different early proteins (representatives of each early transcription unit) was analyzed by Western blot analysis using Abs specific to indicated viral proteins at 6, 12, and 24 h after infection. (B) The relative levels of transcriptional activation were analyzed by real-time RT-PCR analysis of RNA extracted from infected cells collected at 24 h after infection. (C) Role of other mediator subunits. wt MEFs were transfected with pools of control siRNA or siRNA against the indicated mediator subunits. Thirty-six h after transfection, cells were infected with HAdv5-13S and expression of E2-DBP and E4-Orf4 regions was analyzed by real-time RT-PCR at 12 h after infection. The results are expressed relative to cells transfected with control siRNA.

Fig 4

Interaction of SEC with L-E1A. (A) The protein complexes associated with E1A proteins (Flag tagged) were immunoprecipitated with the Flag Ab from human KB cells infected with HF-12S or HF-13S. The protein blots were probed with Abs specific for various constituents of SEC. (B) The interaction of transfected members (Flag tagged) of SEC with E1A proteins (untagged) were determined by Western blotting using the Flag Ab or E1A Ab (M73). The bands corresponding to L-E1A in the top panels are indicated by stars.

Fig 5

Effect of CDK9 on E1A trans-activation. (A) U2OS cells were transfected with the 6×Gal-luciferase reporter and Gal4-DBD-E1A-CR3 trans-activator construct in the presence of increasing concentrations of CDK9 or CDK9 DN mutant (T186A). The relative luciferase activity (compared to vector and wt CDK9-transfected cells) was determined. (B) HeLa cells were transfected with control or CDK9 siRNA pools and infected with HAdv5-dl312, or HF-13S. The level of CDK9 depletion was determined by Western blotting of RNA polymerase II (total), as well as Ser5- and Ser2-phosphorylated forms of pol II, and actin (left panel). The relative levels of expression of E2-DBP and E4-Orf4 regions were determined (right panel). (C) HeLa cells were infected with HAdv5-dl312, HAdv5-12S, or HAdv5-13S (25 PFU/cell) and treated with DMSO (vehicle) or CDK9 inhibitor II (30 μM) at 1 h postinfection with one change of drugs at 6 h postinfection. The effect on phosphorylation was determined using RNA pol II Abs H14 and H5 (left panel). The expression of E2-DBP and E4-Orf4 regions was determined by real-time RT-PCR at 12 h postinfection, and the relative levels were expressed compared to cells infected with HAdv5-dl312 that were treated with the vehicle (DMSO) (right panel). (D) Effect of CDK9 inhibitor on HAdv5 replication. HeLa cells were infected with HAdv5-13S at 5 PFU/cell and treated with DMSO or CDK9 inhibitor II (30 μM) at 1 h postinfection with one change of drugs at 24 h postinfection. The virus yield at 36 and 48 h postinfection was determined by plaque assay on human 293 cells.

Fig 6

Effect of MED23 and MED26 on promoter localization of SEC and TBP. HeLa cells were transfected with control, MED23, or MED26 siRNA pools and infected with 25 PFU/cell HAdv5, HF-12S, or HF-13S. The chromatin was prepared, the transcription factor occupancy was determined by ChIP analysis using Abs specific for RNA polymerase II, E1A (Flag), TBP, AFF4, and CDK9, and the relative occupancy was determined using primers specific to indicated promoter regions. The relative levels of depletion of MED23 and MED26 were determined by RT-PCR analysis and are shown at the bottom of the panel.

Fig 7

Model for transcriptional activation of HAdv early promoters by L-E1A. Based on our results, we suggest that the L-E1A CR3 trans-activation region targets the mediator complex to the viral early promoters through the mediator subunit MED23. The mediator complex is proposed to activate early gene transcription by targeting SEC (bottom), TBP/TFIID (top), and possibly other enhancer binding factors (EBF) to viral early promoters.