Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

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.

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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

Journal of Virology ◽

10.1128/jvi.78.12.6344-6359.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6344-6359 ◽

Cited By ~ 156

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.

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Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.

Journal of Virology ◽

10.1128/jvi.71.10.7361-7371.1997 ◽

1997 ◽

Vol 71 (10) ◽

pp. 7361-7371 ◽

Cited By ~ 38

Author(s):

D M Kube ◽

S Ponnazhagan ◽

A Srivastava

Keyword(s):

Growth Inhibition ◽

Virus Type ◽

Human Cells ◽

Wild Type ◽

Adeno Associated Virus ◽

Rep Proteins ◽

Primary Human Cells

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

Journal of Virology ◽

10.1128/jvi.75.20.9991-9994.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9991-9994 ◽

Cited By ~ 35

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.

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

Viruses ◽

10.3390/v11010038 ◽

2019 ◽

Vol 11 (1) ◽

pp. 38 ◽

Cited By ~ 7

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.

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

International Journal of Oncology ◽

10.3892/ijo.20.6.1211 ◽

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

Journal of Virology ◽

10.1128/jvi.74.24.11511-11521.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11511-11521 ◽

Cited By ~ 29

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.

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Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

Journal of Virology ◽

10.1128/jvi.72.7.5472-5480.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5472-5480 ◽

Cited By ~ 50

Author(s):

Xu-Shan Wang ◽

Benjawan Khuntirat ◽

Keyun Qing ◽

Selvarangan Ponnazhagan ◽

Dagmar M. Kube ◽

...

Keyword(s):

Virus Type ◽

Viral Vector ◽

Nucleotide Sequencing ◽

Wild Type ◽

Helper Plasmid ◽

Adeno Associated Virus ◽

Recombinant Vector ◽

293 Cells ◽

Hairpin Structures

ABSTRACT The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

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Adenovirus-Facilitated Nuclear Translocation of Adeno-Associated Virus Type 2

Journal of Virology ◽

10.1128/jvi.76.22.11505-11517.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11505-11517 ◽

Cited By ~ 89

Author(s):

Wu Xiao ◽

Kenneth H. Warrington ◽

Patrick Hearing ◽

Jeffrey Hughes ◽

Nicholas Muzyczka

Keyword(s):

Virus Type ◽

Dna Hybridization ◽

Nuclear Translocation ◽

Subcellular Fractionation ◽

Nuclear Pore ◽

Wild Type ◽

Nuclear Entry ◽

Adeno Associated Virus ◽

Early Endosomes

ABSTRACT We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t 1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex inhibitor thapsigargan. The rapid nuclear translocation of intact AAV capsids in the presence of Ad suggested that one or more Ad capsid proteins might be altering trafficking. Indeed, coinfection with empty Ad capsids also resulted in the appearance of AAV DNA in nuclei within 40 min. Escape from early endosomes did not seem to be affected by Ad coinfection.

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