Factors that bind to adeno-associated virus terminal repeats

Im, Dong-Soo; Muzyczka, Nicholas

doi:10.1128/jvi.63.7.3095-3104.1989

Cited by 151 publications

(135 citation statements)

References 37 publications

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“…In addition, in a supercoiled plasmid substrate, this modified ITR sequence has the potential to form a T-shaped hairpin structure with contiguous D-A elements, the natural substrate for binding and nicking of AAV Rep proteins ( Fig. 1B) (13,14,23,33). This contiguous D-A stem structure, which is present in the hairpin structure of AAV replication monomer intermediates, as well as the resolved plasmid product described above (Fig.…”

Section: Discussionmentioning

confidence: 99%

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

Xiao

et al. 1997

J Virol

136

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Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

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Section: Discussionmentioning

confidence: 99%

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

Xiao

et al. 1997

J Virol

136

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“…The AAV Rep proteins have multiple functions and are involved in both the replication and gene regulation of the virus. Several of the known properties of the Rep proteins, such as the binding to the ITR DNA, the ATP-dependent site-specific endonuclease, and the DNA helicase activities, are important for its replication function (21)(22)(23)49). The Rep proteins also contain a consensus nucleoside triphosphate (NTP)-binding motif which is involved in the endonuclease, helicase, and ATPase activities of Rep78 and Rep68 (10,12,34,42,57).…”

mentioning

confidence: 99%

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

Kyöstiö

Wonderling

Owens

1995

J Virol

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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.

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“…Proteins were electrophoretically transferred to nitrocellulose membranes in transfer buffer (25 mM Tris-HCl and 192 mM glycine in 20% methanol). MBPRep78 was detected with monoclonal antibody to Rep78 (37). The antibody was detected by chemiluminescence (Amersham).…”

Section: Methodsmentioning

confidence: 99%

“…EMSA. EMSA was conducted as described previously, with minor modi cations (37). 5 0 End-labeled DNA substrate (» 2 ng) was incubated with binding buffer [25 mM HEPES-KOH (pH 7.5), 10 mM MgCl 2 , 1 mMdithiothreitol, 2% glycerol, 0.25 l g bovine serum albumin, 50 mM NaCl, 0.01% NP-40, and 1 l g poly(dI-dC)].…”

Section: Methodsmentioning

confidence: 99%

Adeno‐Associated Virus Rep78 Binds to E2‐Responsive Element 1 of Bovine Papillomavirus Type 1

Chon

Rim

1999

IUBMB Life

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Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses. Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect. In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1. Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells. Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1. This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR. Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure.

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Factors that bind to adeno-associated virus terminal repeats

Cited by 151 publications

References 37 publications

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

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

Adeno‐Associated Virus Rep78 Binds to E2‐Responsive Element 1 of Bovine Papillomavirus Type 1

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