Infectious Molecular Clones of Adeno-Associated Virus Isolated Directly from Human Tissues

ABSTRACT Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

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Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues

Journal of Virology ◽

10.1128/jvi.79.23.14793-14803.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14793-14803 ◽

Cited By ~ 128

Author(s):

Bruce C. Schnepp ◽

Ryan L. Jensen ◽

Chun-Liang Chen ◽

Philip R. Johnson ◽

K. Reed Clark

Keyword(s):

Cultured Cells ◽

Rolling Circle Amplification ◽

Chromosome 1 ◽

Inverted Terminal Repeat ◽

Human Tissues ◽

Wild Type ◽

Tissue Samples ◽

Adeno Associated Virus ◽

Rolling Circle

ABSTRACT Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

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Molecular Characterization of Adeno-Associated Viruses Infecting Children

Journal of Virology ◽

10.1128/jvi.79.23.14781-14792.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14781-14792 ◽

Cited By ~ 53

Author(s):

Chun-Liang Chen ◽

Ryan L. Jensen ◽

Bruce C. Schnepp ◽

Mary J. Connell ◽

Richard Shell ◽

...

Keyword(s):

Amino Acid ◽

Natural Infection ◽

Rolling Circle Amplification ◽

Human Tissues ◽

Degenerate Primers ◽

Rolling Circle ◽

Heparin Sulfate ◽

Capsid Sequence ◽

Arginine Residues

ABSTRACT Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (∼98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

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Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo

Journal of Virology ◽

10.1128/jvi.74.20.9451-9463.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9451-9463 ◽

Cited By ~ 144

Author(s):

Hiroyuki Nakai ◽

Theresa A. Storm ◽

Mark A. Kay

Keyword(s):

Rolling Circle Replication ◽

Chromosomal Dna ◽

Adeno Associated Virus ◽

Rolling Circle ◽

Steady State Levels ◽

Slow Rise ◽

Mouse Hepatocytes ◽

Enzyme Recognition

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

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Consequences of DNA-Dependent Protein Kinase Catalytic Subunit Deficiency on Recombinant Adeno-Associated Virus Genome Circularization and Heterodimerization in Muscle Tissue

Journal of Virology ◽

10.1128/jvi.77.8.4751-4759.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4751-4759 ◽

Cited By ~ 37

Author(s):

Dongsheng Duan ◽

Yongping Yue ◽

John F. Engelhardt

Keyword(s):

Protein Kinase ◽

Muscle Tissue ◽

Southern Blotting ◽

Catalytic Subunit ◽

Inverted Terminal Repeat ◽

Wild Type ◽

Dependent Protein Kinase ◽

Adeno Associated Virus ◽

Dependent Protein

ABSTRACT Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a ∼50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated β-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.

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Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer

Molecules ◽

10.3390/molecules23071833 ◽

2018 ◽

Vol 23 (7) ◽

pp. 1833 ◽

Cited By ~ 2

Author(s):

Kyoung-Ran Kim ◽

Pascal Röthlisberger ◽

Seong Kang ◽

Kihwan Nam ◽

Sangyoup Lee ◽

...

Keyword(s):

Drug Delivery ◽

Cellular Uptake ◽

Rolling Circle Amplification ◽

Modified Nucleotides ◽

Rolling Circle ◽

Dna Nanoparticles ◽

Nuclease Resistance ◽

Cellular Uptake Efficiency

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.

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Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta)

Journal of General Virology ◽

10.1099/vir.0.045948-0 ◽

2012 ◽

Vol 93 (12) ◽

pp. 2668-2681 ◽

Cited By ~ 126

Author(s):

Karyna Rosario ◽

Anisha Dayaram ◽

Milen Marinov ◽

Jessica Ware ◽

Simona Kraberger ◽

...

Keyword(s):

Rolling Circle Amplification ◽

Molecular Techniques ◽

Limited Information ◽

Dna Viruses ◽

Rolling Circle ◽

Viral Genomes ◽

Initiator Protein ◽

Circular Ssdna ◽

Insect Populations ◽

Ssdna Viruses

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

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Herpes Simplex Virus Type 1 Co-Infection Leads to the Formation of Rolling Circle Amplification-Like Adeno-Associated Virus DNA Replication Products

10.1101/2020.12.23.424160 ◽

2020 ◽

Author(s):

Anita F. Meier ◽

Kurt Tobler ◽

Remo Leisi ◽

Anouk Lkharrazi ◽

Carlos Ros ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Rolling Circle Amplification ◽

Helper Virus ◽

Genome Replication ◽

Adeno Associated Virus ◽

Rolling Circle ◽

Simplex Virus ◽

Hsv 1

ABSTRACTAdeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. We demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in presence of HSV-1, and thus provide evidence for an unequivocal rolling circle amplification (RCA) mechanism. This study reveals the ability of AAV to modify the canonical rolling hairpin replication mechanism and to mimic the replication strategy of a co-infecting herpesvirus. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus. Furthermore, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail.

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Concatamerization of Adeno-Associated Virus Circular Genomes Occurs through Intermolecular Recombination

Journal of Virology ◽

10.1128/jvi.73.11.9468-9477.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9468-9477 ◽

Cited By ~ 107

Author(s):

Jusan Yang ◽

Weihong Zhou ◽

Yulong Zhang ◽

Terese Zidon ◽

Terry Ritchie ◽

...

Keyword(s):

Fluorescent Protein ◽

Shuttle Vector ◽

Adeno Associated Virus ◽

Viral Genomes ◽

Large Gene ◽

Tibialis Muscle ◽

Promoter Gene ◽

Infected Muscle ◽

Green Fluorescent

ABSTRACT Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.

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Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome

PLoS Pathogens ◽

10.1371/journal.ppat.1009638 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009638

Author(s):

Anita Felicitas Meier ◽

Kurt Tobler ◽

Remo Leisi ◽

Anouk Lkharrazi ◽

Carlos Ros ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Helper Virus ◽

Inverted Terminal Repeat ◽

Genome Replication ◽

Rolling Circle Replication ◽

Adeno Associated Virus ◽

Rolling Circle ◽

Simplex Virus ◽

Hsv 1

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

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Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

10.26434/chemrxiv.5662315.v1 ◽

2017 ◽

Author(s):

Bo Tian ◽

Peter Svedlindh ◽

Mattias Strömberg ◽

Erik Wetterskog

Keyword(s):

Magnetic Nanoparticles ◽

Ferromagnetic Resonance ◽

Limit Of Detection ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Resonance Field ◽

Isothermal Amplification ◽

Detection Sensitivity ◽

Serum Samples ◽

Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg2P2O7 (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.

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