Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
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.
J Virol. 1995 Nov;69(11):6787-96. Unique Identifier : AIDSLINE MED/96013773 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.
Abstract:
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.
Keywords: 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 960228
M9620404
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Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
