NEGATIVE REGULATION OF THE ADENOASSOCIATED VIRUS (AAV) P-5 PROMOTER INVOLVES BOTH THE P-5 REP BINDING-SITE AND THE CONSENSUS ATP-BINDING MOTIF OF THE AAV REP68 PROTEIN

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 ne gative cis element of the P-5 prompter between the P-5 TATA box and tr anscription start site by using 5' and 3' deletions of the P-5 promote r fused to the chloramphenicol acetyltransferase gene, This element co ntains four imperfect GAGC repeats similar to the Rep recognition sequ ences (RRSs) in the AAV inverted terminal repeats and in the AAV prefe rred integration locus in chromosome 19. Band shift analyses showed th at human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, f ormed 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 guanin e 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 a ccumulation of P-5 transcripts in vivo, In contrast, the Rep68/K340H m utant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negat ive regulation of P-5 transcript levels by Rep68; one involves Rep68 b inding to the P-5 RRS, and another requires the region of Rep68 contai ning the consensus NTP-binding motif, Furthermore, our studies of AAV genomes containing mutated RRS- and/or YY1-binding elements suggest th at transcription factor YY1 binding to the transcription start site of P-5 interferes with Rep68 repression of the P-5 promoter.