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 Novel cis-Acting Replication Element in the Adeno-Associated Virus Type 2 Genome Is Involved in Amplification of Integrated rep-cap Sequences

2001 ◽  
Vol 75 (20) ◽  
pp. 9991-9994 ◽  
Author(s):  
Pascale Nony ◽  
Jacques Tessier ◽  
Gilliane Chadeuf ◽  
Peter Ward ◽  
Aurélie Giraud ◽  
...  
Keyword(s):  
Virus Type ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Cis Acting ◽  
Rep Proteins

ABSTRACT This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

scholarly journals The Adeno-Associated Virus Type 2 Rep Protein Regulates RNA Processing via Interaction with the Transcription Template

2002 ◽  
Vol 22 (11) ◽  
pp. 3639-3652 ◽  
Author(s):  
Jianming Qiu ◽  
David J. Pintel
Keyword(s):  
Rna Polymerase Ii ◽  
Virus Type ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Transcription Unit ◽  
Rna Pol Ii ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins

ABSTRACT The adeno-associated virus type 2 (AAV) large Rep proteins can act to increase the ratio of spliced to unspliced AAV RNA when they are targeted to the transcription template via a Rep binding element. The required Rep binding site is both location and orientation independent; however, Rep enhancement decreases as the distance between the promoter and the intron of the affected transcription unit increases. Only the AAV intron and an extended polyadenylation site must remain for the AAV transcription unit to manifest responsiveness to Rep. A number of promoters, when driving the AAV capsid gene transcription unit, were responsive to targeted Rep, though to various degrees. Transactivation of transcription initiation is not sufficient for the enhancement of RNA processing, because activation of the P40 transcription unit by other activators targeted to this transcription template did not result in enhancement of the ratio of spliced to unspliced AAV RNA. These results suggest that Rep may act as a trans regulator of RNA processing by modulating such functions coupled to RNA polymerase II (RNA pol II) transcription, perhaps by affecting the composition of the transcription complex either prior to or during elongation. These results reveal another way in which gene expression can be regulated by trans-acting proteins and help explain an important feature of the parvovirus life cycle.

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 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 Adeno-Associated Virus Type 5 Utilizes Alternative Translation Initiation To Encode a Small Rep40-Like Protein

2009 ◽  
Vol 84 (2) ◽  
pp. 1193-1197 ◽  
Author(s):  
K. David Farris ◽  
David J. Pintel
Keyword(s):  
Alternative Splicing ◽  
Virus Type ◽  
Translation Initiation ◽  
Adeno Associated Virus ◽  
C Terminus ◽  
Rep Proteins ◽  
Alternative Translation ◽  
Internal Initiation ◽  
Internal Translation

ABSTRACT Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event.

Sign in / Sign up

Export Citation Format

Share Document