scholarly journals Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis-Acting Element outside of the Terminal Repeats and a Minimal Size

2000 ◽  
Vol 74 (24) ◽  
pp. 11511-11521 ◽  
Author(s):  
Gregory E. Tullis ◽  
Thomas Shenk
Keyword(s):  
Virus Type ◽  
Efficient Production ◽  
Replicative Form ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Cis Acting ◽  
Efficient Replication ◽  
Terminal Repeats

ABSTRACT Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence of a positive-acting cis element between nucleotides 194 and 1882 of AAV2. Recombinant AAV2 genomes lacking this region accumulated 14-fold less double-stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required for efficient production of single-stranded viral DNA. Relatively small recombinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less single-stranded DNA per monomer-length replicative-form DNA molecule than wild-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4,712 bp) accumulated similar amounts of single-stranded DNA per monomer-length replicative-form DNA compared to wild-type AAV2. Analysis of two recombinant AAV2 genomes less than 3.5 kb in size indicated that they were deficient in the production of the extended form of monomer-length replicative-form DNA, which is thought to be the immediate precursor to single-stranded AAV2 DNA.

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.

Induction of an Antitumoral Immune Response by Wild-Type Adeno-Associated Virus Type 2 in an In Vivo Model of Pancreatic Carcinoma

Pancreas ◽  
2007 ◽  
Vol 35 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Sven Eisold ◽  
Jan Schmidt ◽  
Eduard Ryschich ◽  
Michael Gock ◽  
Ernst Klar ◽  
...  
Keyword(s):  
Immune Response ◽  
Pancreatic Carcinoma ◽  
Virus Type ◽  
In Vivo Model ◽  
Wild Type ◽  
Adeno Associated Virus

scholarly journals Presence of atrs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

2015 ◽  
Vol 89 (14) ◽  
pp. 7428-7432 ◽  
Author(s):  
Karl Petri ◽  
Richard Gabriel ◽  
Leticia Agundez ◽  
Raffaele Fronza ◽  
Saira Afzal ◽  
...  
Keyword(s):  
Virus Type ◽  
Integration Site ◽  
Dermal Fibroblasts ◽  
Wild Type ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Electrophoretic Mobility Shift Assays ◽  
First Time

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

scholarly journals Circulating Anti-Wild-Type Adeno-Associated Virus Type 2 (AAV2) Antibodies Inhibit Recombinant AAV2 (rAAV2)-Mediated, but Not rAAV5-Mediated, Gene Transfer in the Brain

2004 ◽  
Vol 78 (12) ◽  
pp. 6344-6359 ◽  
Author(s):  
Carmen S. Peden ◽  
Corinna Burger ◽  
Nicholas Muzyczka ◽  
Ronald J. Mandel
Keyword(s):  
Immune Response ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Natural Infection ◽  
Cell Counting ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Live Virus ◽  
The Brain

ABSTRACT Epidemiological studies report that 80% of the population maintains antibodies (Ab) to wild-type (wt) adeno-associated virus type 2 (AAV2), with 30% expressing neutralizing Ab (NAb). The blood-brain barrier (BBB) provides limited immune privilege to brain parenchyma, and the immune response to recombinant AAV (rAAV) administration in the brain of a naive animal is minimal. However, central nervous system transduction in preimmunized animals remains unstudied. Vector administration may disrupt the BBB sufficiently to promote an immune response in a previously immunized animal. We tested the hypothesis that intracerebral rAAV administration and readministration would not be affected by the presence of circulating Ab to wt AAV2. Rats peripherally immunized with live wt AAV2 and naive controls were tested with single intrastriatal injections of rAAV2 encoding human glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Striatal readministration of rAAV2-GDNF was also tested in preimmunized and naive rats. Finally, serotype specificity of the immunization against wt AAV2 was examined by single injections of rAAV5-GFP. Preimmunization resulted in high levels of circulating NAb and prevented transduction by rAAV2 as assessed by striatal GDNF levels. rAAV2-GFP striatal transduction was also prevented by immunization, while rAAV5-GFP-mediated transduction, as assessed by stereological cell counting, was unaffected. Additionally, inflammatory markers were present in those animals that received repeated administrations of rAAV2, including markers of a cell-mediated immune response and cytotoxic damage. A live virus immunization protocol generated the circulating anti-wt-AAV Ab seen in this experiment, while human titers are commonly acquired via natural infection. Regardless, the data show that the presence of high levels of NAb against wt AAV can reduce rAAV-mediated transduction in the brain and should be accounted for in future experiments utilizing this vector.

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.

scholarly journals Methylation Status of the Adeno-Associated Virus Type 2 (AAV2)

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 38 ◽  
Author(s):  
Renáta Tóth ◽  
István Mészáros ◽  
Daniela Hüser ◽  
Barbara Forró ◽  
Szilvia Marton ◽  
...  
Keyword(s):  
Virus Type ◽  
Viral Genome ◽  
Methylation Status ◽  
Replication Initiation ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Cpg Site ◽  
Cg Dinucleotides

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0–1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host’s chromosomes.

Sensitization of sarcoma cells to doxorubicin treatment by concomitant wild-type adeno-associated virus type 2 (AAV-2) infection

2002 ◽  
Author(s):  
Matthias Schwarzbach ◽  
Sven Eisold ◽  
Tatiana Burguete ◽  
Frank Willeke ◽  
Petra Klein-Bauernschmitt ◽  
...  
Keyword(s):  
Virus Type ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Doxorubicin Treatment ◽  
Sarcoma Cells
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