       Document 0403
 DOCN  M9620403
 TI    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.
 DT    9602
 AU    Kyostio SR; Wonderling RS; Owens RA; Laboratory of Molecular and
       Cellular Biology, National Institute; of Diabetes and Digestive and
       Kidney Diseases, National; Institutes of Health, Bethesda, MD
       20892-0840, USA.
 SO    J Virol. 1995 Nov;69(11):6787-96. Unique Identifier : AIDSLINE
       MED/96013773
 AB    Transcript levels from the P5 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 P5 promoter between the P5 TATA box and
       transcription start site by using 5' and 3' deletions of the P5 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 P5 RRS DNA. Within the P5 RRS,
       mutation of a cytosine at position 273 in the AAV sequence to guanine
       abolished Rep68 binding to the DNA. A mutation in the P5 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 P5 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 P5 transcript levels by Rep68; one involves Rep68 binding
       to the P5 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 P5
       interferes with Rep68 repression of the P5 promoter.
 DE    Adenosine Triphosphate/*METABOLISM  Base Sequence  Binding Sites  Cell
       Line  Chloramphenicol Acetyltransferase  Chromosome Mapping
       *Chromosomes, Human, Pair 19  Consensus Sequence  Dependovirus/*GENETICS
       DNA Mutational Analysis  DNA-Binding Proteins/*METABOLISM  *Gene
       Expression Regulation, Viral  Human  HIV Long Terminal Repeat  Kidney
       Molecular Sequence Data  Plasmids  *Promoter Regions (Genetics)  RNA,
       Messenger/BIOSYNTHESIS  Sequence Deletion  Transcription, Genetic
       Transfection  TATA Box  Viral Proteins/*METABOLISM  *Virus Integration
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

