Cloning, Expression and Purification of Full-Length Rep78 of Adeno-associated Virus as a Fusion Protein with Maltose Binding Protein in Escherichia coli

Batchu, Ramesh B.; Miles, Dorothy A.; Rechtin, Tammy M.; Drake, Richard; Hermonat, Paul L.

doi:10.1006/bbrc.1995.1396

Cited by 11 publications

(13 citation statements)

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“…1-3 intact. The reconstituted wild-type and mutant AAV genomes All of the completed TRs contained TRI, which was used in previous studies [19,29]. Each completed mutant TR was composed of TR1 plus the appropriately named TR5 and TR6 oligonucleotides.…”

Section: Resultsmentioning

confidence: 99%

“…Each mutant TR was composed of TRI plus the appropriately named TR5 and TR6 oligonucleotides. All of the TRs contained TRI, the largest and most expensive oligonucleotide, which has been used in previous studies [19,29]. The TR1 oligonucleotide was ligated with the appropriate set of TR5 and TR6 onto 300 ng of the BssHII isolated 4.5 kb DNA fragment at a ratio of one AAV genomic terminus to three of each oligonucleotide to generate the mutant genomes.…”

Section: Construction Of Aav Tr Mutant Dna Genomesmentioning

confidence: 99%

“…Of the several related proteins produced from the AAV rep gene, the Rep68/78 proteins (the full length ORF products; Rep68 is produced by a splicing event at its 3' end) are the most important viral proteins involved in AAV DNA replication [11,12]. Rep78 is a multifunctional protein which binds DNA, binds ATP, has DNA helicase activity, and has DNA endonuclease activity [13][14][15][16][17][18][19][20]. The Rep78 protein recognizes and interacts with at least three regions within the AAV TR DNA.…”

Section: Introductionmentioning

confidence: 99%

“…The DNA sequence which constitutes the major binding site for the Rep78 protein, as determined by DNAse I footprint analysis and by electrophoretic mobility shift assays (EMSA) with oligonucleotides, is a triplex concatemeric sequence of the motif 5' GAGC 3' (or GCTC) [16,19]. A variety of EMSA studies demonstrate that three such contiguous motifs, with appropriate adjacent sequences, allow for very strong binding by Rep78 [21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

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Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

et al. 1996

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The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC 3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.

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Section: Resultsmentioning

confidence: 99%

Section: Construction Of Aav Tr Mutant Dna Genomesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

et al. 1996

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“…It has been noticed by others [11] that the cis TAR sequence of HIV-1, to which the HIV-1 encoded tat trans-activator binds as RNA, contains multiple GCTC motifs [30,31]. This laboratory has been studying Rep78 functions utilizing a maltose binding protein-Rep78 chimeric (MBP-Rep78), produced in bacteria, which has all the known biochemical functions of wild type Rep78 from eukaryotes [32].…”

Section: Introductionmentioning

confidence: 99%

The trans‐inhibitory Rep78 protein of adeno‐associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat

Batchu

Hermonat

1995

FEBS Letters

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The large rep gene products, Rep78 and Rep68, of adeno-associated virus (AAV) are pleiotropic effector proteins which are required for AAV DNA replication and the transregulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, these rep products are able to inhibit the replication and gene expression of human immunodeficiency virus type 1 (HIV-1) and a number of DNA viruses. Here, it is demonstrated that Rep78, as a chimeric with the maltose binding protein, directly binds the full-length HIV-I long terminal repeat (LTR), and to a subset of these sequences containing the trans-activation response (TAR) sequence as DNA. These interactions, an effector protein physically binding a target promoter, suggest a direct mechanism of action for Rep78 inhibition. Furthermore, competitive binding studies between the TAR region and the full-length HIV-LTR, strongly suggested that another site(s) within the LTR was also bound by Rep78. Finally, as Rep78 binding is also believed to he affected by secondary structure within the DNA, it was found that Rep78 preferentially binds with HIV-LTR sequences with promoted secondary structure generated by heat denaturation and rapid cooling.

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The Adeno-Associated Virus Rep78 Major Regulatory Protein Binds the Cellular TATA-Binding Proteinin Vitroandin Vivo

et al. 1998

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Rep78 is the major regulatory protein of adenoassociated virus (AAV). Rep78 is able to transcriptionally regulate all three of AAV's promoters, as well as a variety of heterologous promoters. In an attempt to understand the mechanism of action by which Rep78 is able to regulate gene expression, we are investigating Rep78's possible protein-protein interaction with basal transcription factors. One such critical basal transcription factor is the human TATA binding protein, TBP. TBP is a core factor required for the assemblage of the transcription initiation complex, TFIID. In this report an in vitro interaction between Rep78 and TBP was demonstrated in three different assay systems, including West(far)-Western analysis, electrophoretic mobility shift assay-supershift, and coimmunoprecipitation. Furthermore, using the yeast GAL4 two-hybrid system, an in vivo interaction between Rep78 and TBP was also demonstrated. Further still, the amino half of Rep78 is shown to be needed for Rep78-TBP interaction. Mutations within this region of Rep78 are known to be defective for transcriptional regulatory ability, suggesting a biological role for this interaction. Thus, Rep78 may regulate transcription through binding and regulating TBP's numerous interactions. Furthermore, as Rep78 is known to bind at least one other transcription factor (Sp 1) and likely others, Rep78 may function as a TBP-associated factor in an altered TFIID-like complex.

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Cloning, Expression and Purification of Full-Length Rep78 of Adeno-associated Virus as a Fusion Protein with Maltose Binding Protein in Escherichia coli

Cited by 11 publications

References 0 publications

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

The trans‐inhibitory Rep78 protein of adeno‐associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat

The Adeno-Associated Virus Rep78 Major Regulatory Protein Binds the Cellular TATA-Binding Proteinin Vitroandin Vivo

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