Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein

McCarty, Doug; Pereira, Daniel J.; Zolotukhin, Irene; Zhou, Xi; Ryan, J H; Muzyczka, Nicholas

doi:10.1128/jvi.68.8.4988-4997.1994

Cited by 143 publications

(113 citation statements)

References 39 publications

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“…Relative affinity of Rep68 to p5, p19, p40, and the TR. We previously demonstrated that purified Rep68 interacts with a linear 22-bp sequence within the TR and the p5 promoter (38). Additionally, there appeared to be weaker interactions with the p19 and p40 promoter regions, and RBE-like sequences were found in both of these promoters (Fig.…”

Section: Resultsmentioning

confidence: 86%

“…In contrast, deletion of nt 780 to 809, including the CATT element (27) (dotted box), is not required for p19 promoter activity. Sequences with homology to the linear TR RBE are underlined by a dotted line (38). The boundary between fragments I and II, which were used in protein binding experiments, is indicated by arrows.…”

Section: Methodsmentioning

confidence: 99%

“…Genetic analysis of the AAV genome has demonstrated that the p5 Rep proteins (Rep78 and Rep68) are required to reg-ulate transcription from all three promoters and to replicate the viral DNA (20,53). Several biochemical properties have been characterized for the p5 Rep proteins during replication; these include site-specific endonuclease (24,51), DNA helicase (24,58), and DNA binding (24,25,38) activities. In the absence of Ad, Rep has been shown to repress both p5 and p19 transcription (21,30).…”

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confidence: 99%

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

Pereira

Muzyczka

1997

J Virol

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Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions ؊50 and ؊130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position ؊140. Mutational analysis of the p19 promoter suggested that the SP1 site at ؊50 and the cAAP site at ؊140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA.

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Section: Resultsmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Pereira

Muzyczka

1997

J Virol

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“…The AAV ITRs can assume a T-shaped structure (43) and exist in two conformations termed "flip" or "flop" and generated during the rolling hairpin mechanism of replication (44). There are three salient features of the AAV ITR that are required for vector production: (i) a 16-nucleotide tetrameric repeat (GAGC) sequence that is specifically bound by the Rep68/78 proteins termed the Rep-binding element (RBE), (ii) a sequence within the ITR internal hairpin that serves to orient Rep68/78 towards its resolution site termed RBE' (45)(46)(47)(48)(49), and (iii) the terminal resolution sequence (trs), which is the site of the strand-specific Rep68/78 endonuclease activity (44,(50)(51)(52). The N-termini of the larger Rep proteins contain a DNA-binding domain, as well as strand-specific endonuclease activity, necessary for initiating replication during virion production and site-specific integration (46,(53)(54)(55)(56).…”

Section: Adeno-associated Virus Genomementioning

confidence: 99%

Adeno-associated Virus as a Mammalian DNA Vector

2015

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In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells.

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“…The site-specific integration of AAV-2 is located in a non-repetitive element at the position 19q13,42 corresponding to the long arm of the chromosome 19, in a gene-dense region named AAVS1 (for AAV integration site 1) ( Figure 3-A) (Kotin et al, 1991). Analysis of AAVS1 host sequence revealed two cis-acting sequences involved in AAV-2 integration: the terminal resolution site (TRS) corresponding to the Rep-specific endonuclease site and the Rep binding site (RBS) (Brister and Muzyczka, 1999, McCarty et al, 1994a, McCarty et al, 1994b. Interestingly, this TRS-RBS motif is also present in the ITR of the viral genome, suggesting that the sequence homology between AAV-2 ITR and the host genome site -TRS and RBS sequencesplays a role in AAV-2 integration.…”

Section: Adeno-associated Virus Type 2 (Aav-2)mentioning

confidence: 99%