scholarly journals Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

2007 ◽  
Vol 81 (8) ◽  
pp. 3721-3730 ◽  
Author(s):  
Mary Murphy ◽  
Janette Gomos-Klein ◽  
Marko Stankic ◽  
Erik Falck-Pedersen
Keyword(s):  
Virus Type ◽  
Transcriptional Control ◽  
Helper Virus ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Rep Proteins

ABSTRACT The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

scholarly journals Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-rep Sequence in cis

2002 ◽  
Vol 76 (11) ◽  
pp. 5411-5421 ◽  
Author(s):  
Nicola J. Philpott ◽  
Catherine Giraud-Wali ◽  
Carolyn Dupuis ◽  
Janette Gomos ◽  
Henry Hamilton ◽  
...  
Keyword(s):  
First Generation ◽  
Promoter Sequence ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Enhancer Element ◽  
Site Specific ◽  
Site Specific Integration ◽  
Gene Therapy Vectors ◽  
A Site

ABSTRACT The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

scholarly journals Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration

2007 ◽  
Vol 82 (5) ◽  
pp. 2590-2593 ◽  
Author(s):  
Mickaël Guilbaud ◽  
Gilliane Chadeuf ◽  
Fabio Avolio ◽  
Achille François ◽  
Philippe Moullier ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Virus Type ◽  
Promoter Region ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Human Chromosome 19

ABSTRACT The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.

scholarly journals Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein

1999 ◽  
Vol 73 (4) ◽  
pp. 2682-2693 ◽  
Author(s):  
Masashi Urabe ◽  
Yoko Hasumi ◽  
Akihiro Kume ◽  
Richard T. Surosky ◽  
Gary J. Kurtzman ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Inverted Terminal Repeat ◽  
Similar Reaction ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Rep Proteins

ABSTRACT The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trsnicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

scholarly journals Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

2000 ◽  
Vol 74 (19) ◽  
pp. 8831-8842 ◽  
Author(s):  
Stefania Lamartina ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site ◽  
First Time ◽  
Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

1994 ◽  
Vol 68 (2) ◽  
pp. 797-804 ◽  
Author(s):  
J A Chiorini ◽  
M D Weitzman ◽  
R A Owens ◽  
E Urcelay ◽  
B Safer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virus Type ◽  
Fusion Proteins ◽  
Biologically Active ◽  
Adeno Associated Virus ◽  
Rep Proteins

scholarly journals Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter

1998 ◽  
Vol 72 (6) ◽  
pp. 4811-4818 ◽  
Author(s):  
Xu-Shan Wang ◽  
Arun Srivastava
Keyword(s):  
Binding Site ◽  
Virus Type ◽  
Hela Cells ◽  
Biologically Active ◽  
Viral Gene ◽  
Adeno Associated Virus ◽  
Autonomous Replication ◽  
293 Cells ◽  
Rep Proteins

ABSTRACT The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.

scholarly journals Adeno-associated virus Type 2 Rep proteins mediate integration of lentiviral vectors

2014 ◽  
Author(s):  
Victor J McAlister ◽  
Anthony T Craig ◽  
Roland A Owens
Keyword(s):  
Virus Type ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Specific Pcr ◽  
Rep Proteins ◽  
Dividing Cells ◽  
Lentivirus Vectors

Aims: Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells, AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to integrate preferentially into a particular locus within human chromosome 19, designated AAVS1(also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this integration. Recent data suggest that Rep68 and Rep78 can mediate integration of non-AAV2 DNA with free ends. To test this hypothesis, we targeted insertion of different lentiviral vectors to AAVS1. Methods: Cells were co-infected with wild-type AAV2, and integrase-proficient or integrase-deficient lentivirus vectors. A highly specific PCR-based assay was used to detect lentivirus integration at AAVS1. Similar experiments were performed using lentiviral vectors containing the AAV2 rep gene. Results: All lentiviral vectors tested integrated at AAVS1, if the rep gene was present either within the lentiviral vector or supplied in trans. All that was required for integration at AAVS1 was the amino acid sequence shared between Rep68 and Rep78. The results were similar with integrase-proficient or integrase-deficient lentiviral vectors. Conclusions. The inclusion of the rep gene with lentiviral vectors may produce more predictable integration patterns.

scholarly journals Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

1999 ◽  
Vol 73 (10) ◽  
pp. 8235-8244 ◽  
Author(s):  
Jianwen Wu ◽  
Michael D. Davis ◽  
Roland A. Owens
Keyword(s):  
Virus Type ◽  
Helicase Activity ◽  
Endonuclease Activity ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Rabbit Reticulocyte Lysate System ◽  
Wide Range

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

Sign in / Sign up

Export Citation Format

Share Document