Studies of the mechanism of transactivation of the adeno-associated virus p19 promoter by Rep protein

Abstract

During adeno-associated virus (AAV) type 2 productive infections, the p19 promoter of AAV is activated by the AAV Rep78 and Rep68 proteins. Rep-induced activation of p19 depends on the presence of one of several redundant Rep binding elements (RBEs) within the p5 promoter or within the terminal repeats (TR). In the absence of the TR, the p5 RBE and the p19 Sp1 site at position -50 are essential for p19 transactivation. To determine how a Rep complex bound at p5 induces transcription at p19, we made a series of p19 promoter chloramphenicol acetyltransferase constructs in which the p5 RBE was inserted at different locations upstream or downstream of the p19 mRNA start site. The RBE acted like a repressor element at most positions in the presence of both Rep and adenovirus (Ad), and the level of repression increased dramatically as the RBE was inserted closer to the p19 promoter. We concluded that the RBE by itself was not a conventional upstream activation signal and instead behaved like a repressor. To understand how the Rep-RBE complex within p5 activated p19, we considered the possibility that its role was to function as an architectural protein whose purpose was to bring other p5 transcriptional elements to the p19 promoter. In order to address this possibility, we replaced both the p5 RBE and the p19 Sp1 site with GAL4 binding sites. The modified GAL4-containing constructs were cotransfected with plasmids that expressed GAL4 fusion proteins capable of interacting through p53 and T-antigen (T-ag) protein domains. In the presence of Ad and the GAL4 fusion proteins, the p19 promoter exhibited strong transcriptional activation that was dependent on both the GAL4 fusion proteins and Ad infection. This suggested that the primary role of the p5 RBE and the p19 Sp1 sites was to act as a scaffold for bringing transcription complexes in the p5 promoter into close proximity with the p19 promoter. Since Rep and Sp1 themselves were not essential for transactivation, we tested mutants within the other p5 transcriptional elements in the context of GAL4-induced looping to determine which of the other p5 elements was necessary for p19 induction. Mutation of the p5 major late-transcription factor site reduced p19 activity but did not eliminate induction in the presence of the GAL4 fusion proteins. However, mutation of the p5 YY1 site at position -60 (YY1-60) eliminated GAL4-induced transactivation. This implicated the YY1-60 protein complexes in p19 induction by Rep. In addition, both basal p19 activity and activity in the presence of Ad increased when the YY1-60 site was mutated even in the absence of Rep or GAL4 fusion proteins. Therefore, there are likely to be alternative p5-p19 interactions that are Rep independent in which the YY1-60 complex inhibits p19 transcription. We concluded that transcriptional control of the p19 promoter was dependent on the formation of complexes between the p5 and p19 promoters and that activation of the p19 promoter depends largely on the ability of Rep and Sp1 to form a scaffold that positions the p5 YY1 complex near the p19 promoter.

FIG. 1.

Sequences of the p5 and p19 promoters. The known transcription elements in the p5 and p19 promoters (6, 24, 36, 38, 40, 45) are diagrammed along with their approximate positions upstream of the mRNA start sites. p5CAT plasmid contains only the p5 promoter elements driving CAT expression, while pIM45CAT3 contains both the p5 and p19 promoter elements, with the p19 promoter driving CAT expression. Dotted lines indicate intervening sequences that are not shown. Italics indicate substitutions within the p19CAT3 promoter at positions −130, −110, and −20 that were previously demonstrated to have little effect on p19 transcription (40) and the sub262 substitution within the p5 RBE that no longer binds Rep protein (36, 38). Boldface type and italics indicate the GAL4 substitutions in 2xGAL4 that replace the p5 RBE and the p19 Sp1-50 sites. A CREB/ATF site, which has been mapped upstream of the MLTF site in p5, is not shown (29).

FIG. 2.

Activity of the wild-type p5 and p19 promoters. (A) Diagram of the p5 and p19 promoter elements in the CAT reporter constructs p5CAT, pIM45CAT3, and psub262CAT3. The asterisk indicates a stop codon in the Rep78 open reading frame at amino acid position 71. (B to D) Graphic representation of CAT activity from the p5CAT, pIM45CAT3, and psub262CAT3 reporter constructs after transfection in A549 cells in the presence of plasmid alone (Mock) or with the addition of 1 μg of pIM45 (Rep), Ad at an MOI of 5 (Ad), or 1 μg of pIM45 and Ad at an MOI of 5 (Ad & Rep). Error bars indicate 1 SD.

FIG. 3.

Effect of RBE position on p19 activity. Shown are diagrams of transcription elements within the wild-type (wt) p5 and p19 promoters (A), of the parental plasmid p19CAT3 (B), and of derivatives of p19CAT3 (C to G), in which an RBE was inserted at various positions with respect to the start of p19 transcription (positions −700 to +1225). The p19CAT3 plasmid has the minimum p19 promoter elements, as shown in Fig. 1, and no p5 promoter elements. The CAT activity of each construct was determined after transfection into A549 cells in the presence of plasmid alone (Mock), pIM45 (Rep), Ad, or pIM45 and Ad (Ad & Rep). Error bars indicate 1 SD.

FIG. 4.

Linearization of pIM45CAT3 plasmid to inhibit readthrough transactivation. The graph shows p19 CAT activity from A549 cells transfected with supercoiled pIM45CAT3 plasmid (A) or plasmid that had been linearized by digestion with NruI (B) or SphI (C). For each case, the activity in the presence of Ad infection at an MOI of 5 (Ad) was compared with the activity in the presence of Ad infection and transfection with 1 μg of pIM45 (Ad & Rep). The values shown for pIM45CAT3 (A) are the same as those shown in Fig. 2C and are presented for comparison. Error bars indicate 1 SD.

FIG. 5.

Effect of poly(A) insertion to inhibit readthrough activation. Shown is a diagram of transcription elements within the wild-type (wt) p5 and p19 promoters (A). A poly(A) sequence was inserted into pIM45CAT3 (C) and psub262CAT3 (E) between the p5 and p19 promoters to determine the effect of a poly(A) signal on p19 activity when the plasmid was transfected by itself (Mock), in the presence of the Rep-expressing plasmid pIM45 (Rep), in the presence of Ad at an MOI of 5 (Ad), or in the presence of both pIM45 and Ad (Rep & Ad). The asterisks indicate that the Rep coding sequence has a stop codon at amino acid position 71 of Rep78 to prevent Rep expression from the CAT reporter plasmids. The values shown for pIM45CAT3 (B) and psub262CAT3 (D) are the same as those shown in Fig. 2C and D and are presented here for comparison. Error bars indicate 1 SD.

FIG. 6.

Replacement of Rep and Sp1 with hybrid GAL4 factors. (A) Diagram illustrating that the RBE in the p5 promoter and the Sp1-50 site in the p19 promoter of pIM45CAT were both replaced with GAL4 binding sites. See Fig. 1 for the specific sequence modifications. In the presence of hybrid GAL4-p53 and GAL4-T-ag fusion proteins, the remaining p5 promoter elements and their associated protein complexes would be brought into close proximity with the p19 promoter, resulting in a DNA loop structure. (B) Transactivation of the pIM45CAT3 reporter plasmid when transfected alone (Mock) or with 1 μg of pIM45 plasmid (Rep), with Ad at an MOI of 5 (Ad), or Ad at an MOI of 5 plus pIM45 transfection (Ad & Rep). The data shown are identical to those shown in Fig. 2B and are presented here for comparison. (C) Transactivation of the 2xGAL plasmid when transfected alone (Mock) or with 1 μg of pM-53 plasmid (p53), with 1 μg of pM-Tg plasmid (T-Ag), with 1 μg of both pM-53 and pM-Tg plasmid (p53 & T-Ag), with Ad at an MOI of 5 (Ad), with Ad at an MOI of 5 and the two-hybrid plasmids (Ad, p53, T-Ag), or with Ad at an MOI of 5 and either 1 μg of pM-53 (Ad & p53) or 1 μg of pM-Tg (Ad & T-Ag). Error bars indicate 1 SD.

FIG. 7.

Transactivation of the 2xGAL p19 promoter by mutant p5 promoters. The 2xGAL plasmid containing a wild-type or mutant p5 promoter was transfected alone (Mock) or with 1 μg each of the hybrid plasmids expressing GAL4-p53 and GAL4-T-ag, pM-53 and pM-Tg (Hybrid); with Ad at an MOI of 5 (Ad); or with Ad at an MOI of 5 and the hybrid GAL4-expressing plasmids (Ad & Hybrid). See Fig. 1 for the specific sequence modifications. (A) Transactivation of the 2xGAL4 plasmid containing a wild-type p5 promoter. These values are extracted from those shown in Fig. 6B and are presented here for comparison. (B) Transactivation of the 2xGAL reporter construct lacking the p5 promoter MLTF binding site. (C) Transactivation of the 2xGAL reporter construct lacking the p5 promoter YY1-60 binding site. (D) Transactivation of the 2xGAL reporter construct lacking the p5 promoter MLTF and YY1-60 binding sites. (E) Transactivation of the 2xGAL reporter construct lacking the p5 promoter MLTF, YY1-60, and YY1+1 binding sites. Error bars indicate 1 SD.

FIG. 8.

Model of p19 promoter transactivation by interaction with transcription factor complexes bound to p5.