Negative regulation of the adeno-associated virus (AAV) P5 promoter involves both the P5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein

Abstract

Transcript levels from the P 5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P 5 promoter between the P 5 TATA box and transcription start site by using 5 and 3 deletions of the P 5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P 5 RRS DNA. Within the P 5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P 5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P 5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P 5 transcript levels by Rep68; one involves Rep68 binding to the P 5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS-and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P 5 interferes with Rep68 repression of the P 5 promoter.

The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection

“…2A, pIM45). This result was consistent with several reports that demonstrate Rep repression of p5 in the absence of helper virus (19,25,26). When p5CAT-transfected cells were infected with Ad, a 12.5-fold increase in CAT activity was observed ( Fig.…”

“…Rep proteins negatively regulate p5CAT activity in the presence and absence of an Ad coinfection. As mentioned earlier, several groups have reported that in the absence of Ad helper virus, the Rep proteins negatively regulate the p5 promoter (19,25,26).In contrast, it has also been reported that in the presence of Ad, Rep can both transactivate and repress p5 transcription (3,27). To resolve Rep action at p5, expression of a CAT reporter under p5 control was used to assay promoter activity.…”

“…1A) (9,41). It has been proposed that Rep negatively regulates p5 by inhibiting YY1 from binding to p5 (26). To provide evidence that the function of Rep repression was to counter E1a activity, we tested the ability of Rep to counteract the transactivation properties of the Ad E1 region by using p5CAT as the reporter.…”

“…In the absence of helper virus, all four Rep proteins have been observed to repress p5 and p19 transcription (19,25). Kyostio et al have shown that repression at p5 is mediated in part by Rep binding to the p5 RBE (26). In addition, they proposed that the consensus ATP binding region within Rep contributes to the ability of Rep to repress transcription.…”

The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter

“…In the absence of Ad, Rep has been shown to repress both p5 and p19 transcription (21,30). Repression of p5 is mediated in part by direct binding of Rep78 or Rep68 to the p5 Rep binding element (RBE) (31,43). In the presence of helper virus, Rep78/68 transactivation of the p19 and p40 promoters is dependent on the presence of the RBEs in the p5 promoter and the terminal repeat (TR) (36,43).…”