Comparison of E1A CR3-dependent transcriptional activation across six different human adenovirus subgroups

Abstract

The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.

FIG. 1.

Transcriptional activation by E1A CR3s from six human adenovirus subgroups. (A) Sequence alignment of human adenovirus E1A CR3 fragments used from types Ad12 (subgroup A), Ad3 (subgroup B), Ad5 (subgroup C), Ad9 (subgroup D), Ad4 (subgroup E), and Ad40 (subgroup F). Asterisks indicated zinc-coordinating cysteines. Arrows indicate residues in Ad9 E1A CR3 that differ from those in Ad5 E1A CR3 and that are critical for transcriptional activation. (B) Intrinsic transcriptional activation properties of representative E1A CR3s. A549, HeLa, C33A, or HT1080 cells or MEFs were cotransfected with a Gal4-responsive luciferase reporter and vectors expressing the indicated Gal4-DBD fusions. Luciferase activity is expressed as a percentage of the Ad5 CR3 fold activation above that for the empty vector ± SD.

FIG. 2.

Squelching of Ad5 E1A CR3 function. (A) Design and titration of squelching assay. A549 cells were cotransfected with a Gal4-responsive luciferase reporter, vectors expressing Gal4 alone, or Gal4-Ad5 CR3 as activators and either EGFP or increasing amounts (0 ng, 20 ng, 100 ng, 500 ng, and 1,000 ng) of vector expressing the EGFP-Ad5 CR3 fusion as a competitor. Luciferase activity is expressed as a percentage of the fold activation of Gal4-Ad5 CR3 above that for the vector alone with EGFP as the competitor ± SD. (B) Activation of established mutants of E1A CR3 as Gal4 fusions. A549 cells were cotransfected with a Gal4-responsive luciferase reporter and vectors expressing the indicated Gal4 fusions to E1A CR3 or empty vector. Luciferase activity is expressed as a percentage of the fold activation above that for the vector ± SD. (C) Interaction of mutants of Ad5 E1A CR3 with known cellular targets of CR3. E1A CR3 fusions were immunoprecipitated with anti-myc antibody and blotted with anti-HA antibody for the indicated targets. Inputs are probed with anti-myc antibody for CR3s or anti-HA antibody for MED23, TBP, hSug1, and p300. IP, immunoprecipitation. (D) Squelching of E1A CR3-dependent transactivation by Ad5 CR3 mutants. A549 cells were cotransfected as described for panel A but with equal amounts of vectors expressing EGFP fused to the indicated Ad5 CR3 mutant or HPV E7 (1,000 ng). (E) Sequestration of hSug1 is not responsible for transcriptional squelching by CR3 of the V147L mutant. Human A549 cells were cotransfected as described for panel D with either empty vector (pcDNA4) or a hSug1 expression vector (pcDNA4-hSug1) in a 1:1:1:1 ratio. The levels of activation in vector-transfected cells versus those in hSug1-transfected cells were compared by Student's t test, and P values are indicated above the bars.

FIG. 3.

Squelching of Ad5 E1A CR3-dependent activation by the CR3 domains from representative human adenovirus types. (A) Human A549 cells were cotransfected with a Gal4-responsive luciferase reporter, vectors expressing Gal4 alone, or Gal4-Ad5 CR3 as the activator and either EGFP or EGFP fused to each of the indicated E1A CR3s as the competitor. Luciferase activity is expressed as a percentage of the fold activation of Gal4-Ad5 CR3 above that for the vector alone with EGFP as the competitor ± SD. (B) Summary of transcriptional properties of representative E1A CR3s. The transcriptional activation, squelching, and expression levels of each E1A CR3 are summarized relative to those of Ad5 E1A CR3.

FIG. 4.

MED23 is targeted by E1A CR3s from multiple Ad types. (A) Coimmunoprecipitation of MED23 and the 13S mRNA-encoded E1A proteins from each human adenovirus subgroup. Human HT1080 cells were cotransfected with a vector expressing HA-tagged human MED23 and vectors expressing either EGFP or an EGFP fused to the indicated E1A proteins. E1A proteins were immunoprecipitated with anti-EGFP antibody and blotted with anti-HA antibody. Inputs were probed with EGFP antibody for E1A proteins or HA antibody for MED23. (B) Coimmunoprecipitation of MED23 with the E1A CR3 domains from each human Ad subgroup. Human HT1080 cells were cotransfected with vectors expressing HA-tagged human MED23 and either myc-EGFP or myc-EGFP fused to the indicated E1A CR3 domain. E1A CR3 domains were immunoprecipitated with anti-myc antibody and blotted with anti-HA antibody. Inputs were probed with anti-myc antibody for CR3s or anti-HA antibody for MED23. (C) E1A CR3 activation in MED23-null MEFs. MED23-null MEFs and wt littermate-derived MEFs were cotransfected with a Gal4-responsive luciferase reporter and vectors expressing Gal4 fused to the indicated E1A CR3 domain (white and black bars, respectively). MED23-null MEFs were also transfected with the Gal4-responsive luciferase reporter, vectors expressing the indicated Gal4-E1A CR3 domain fusions, and an expression vector for human MED23 (gray bars). Luciferase activity is expressed as the fold activation over that for Gal4-DBD alone ± SD.

FIG. 5.

TBP is a conserved cellular target of E1A CR3 from multiple Ad types. (A) Coimmunoprecipitation of TBP with representative E1A CR3s. HT1080 cells were cotransfected with a vector expressing HA-tagged TBP and vectors expressing the indicated E1A CR3s fused to EGFP. E1A CR3s were immunoprecipitated with 9E10 antibody and blotted for HA. (B) Negative coimmunoprecipitation of pRb with representative E1A CR3s. HT1080 cells were cotransfected with a vector expressing the indicated E1A CR3s fused to EGFP or genomic E1A. E1A CR3s were immunoprecipitated with anti-myc antibody, and E1A proteins were immunoprecipitated with M73 and blotted for endogenous pRb. (C) siRNA knockdown of TBP and the effect on transcriptional activation by E1A CR3. HeLa cells were transfected with 5 nM siRNA (negative control or TBP specific) and at 2 days after siRNA transfection were retransfected with a Gal4-reponsive luciferase reporter and an expression vector for the indicated Gal4-CR3 fusions. At 48 h posttransfection (120 h after siRNA transfection), cells were harvested and assayed for luciferase activity. Luciferase activity is expressed as the fold above that for Gal4-DBD alone ± SD. The levels of activation in control versus TBP-knockdown cells were compared by Student's t test, and P values are indicated. (Inset) Levels of TBP in knockdown and control cells.

FIG. 6.

Human Sug1 is a conserved target of E1A CR3 from multiple Ad types. (A) Coimmunoprecipitation of hSug1 with representative Ad E1A CR3s. HT1080 cells were cotransfected with pcDNA4HA-hSug1 and vectors expressing the indicated E1A CR3s fused to myc-EGFP. E1A CR3s were coimmunoprecipitated with anti-myc antibody and blotted for HA. (B) siRNA knockdown of hSug1 and the effect on transcriptional activation by E1A CR3. HeLa cells were transfected with 5 nM siRNA (negative control or Sug1 specific) and at 2 days posttransfection were retransfected with a Gal4-reponsive luciferase reporter and an expression vector for the indicated Gal4-CR3 fusions. At 48 h posttransfection (120 h after siRNA transfection), cells were harvested and assayed for luciferase activity. Luciferase activity is expressed as the fold above that for Gal4-DBD alone ± SD. The levels of fold activation of control versus hSug1 siRNA-treated cells were compared by Student's t test, and P values are indicated above the bars. (Inset) Levels of hSug1 in knockdown and control cells.

FIG. 7.

p300 is a conserved target of E1A CR3s from multiple Ad types. (A) Interaction of p300 with the E1A CR3 domains of different Ad types. Human HT1080 cells were cotransfected with an expression vector for HA-tagged p300 and expression vectors for the indicated E1A CR3 myc-EGFP fusions. EGFP fusions were immunoprecipitated with a cocktail of anti-myc and anti-GFP antibodies and blotted with HA. (B) Effect of siRNA depletion of p300 on E1A CR3-dependent activation. HeLa cells were transfected with 20 nM custom siRNA directed against p300 or control siRNA and at 3 days posttransfection were retransfected with a Gal4-reponsive luciferase reporter and an expression vector for the indicated Gal4-CR3 fusions. At 48 h posttransfection (120 h after siRNA transfection), cells were harvested and assayed for luciferase activity. Luciferase activity is expressed as the fold above that for Gal4-DBD alone ± SD. The levels of fold activation of control versus p300 siRNA-treated cells were compared by Student's t test, and P values are indicated above the bars. (Inset) Levels of p300 in knockdown and control cells.

FIG. 8.

Transactivation by full-length E1A. (A) Transactivation by full-length E1A proteins in the context of chromatin. U2OS-UAS cells, which contain an integrated Gal4-responsive luciferase reporter, were transfected with expression vectors for either the indicated E1A CR3 domains or the indicated full-length 13S E1A proteins. Luciferase activity is expressed as the fold activation above that for Gal4 alone ± SD. (B) Transactivation of the Ad5 and Ad9 E4 promoters during infection. At 16 h postinfection with the indicated viruses, the expression level of E4orf6/7 mRNA was determined by qRT-PCR. The expression level of E4orf6/7 relative to the levels for GAPDH and E1A is indicated, and the expression level for cells infected with dl312 was set equal to 1.