Separation of Two Types of Adeno-Associated Virus Particles Containing Complementary Polynucleotide Chains

Berns, Kenneth I.; Adler, Stuart P.

doi:10.1128/jvi.9.2.394-396.1972

Cited by 84 publications

(25 citation statements)

References 7 publications

(7 reference statements)

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“…In this assay, psub201 DNA was the most efficient helper (5 x 105 hygromycin-resistant colonies per ml of lysate). pAAV/no-tr was very inefficient (3 x 102 hygromycin-resistant colonies per ml of lysate), while pAAV/Ad performed at an intermediate level (3 x 104 hygromycinresistant colonies per ml of lysate). Cotransfection with pAAV/Ad DNA generated a yield of AAV/hyg reduced by a factor of 10 to 20 as compared with stocks generated by cotransfection with psub201 DNA.…”

Section: Ap Asmidsmentioning

confidence: 99%

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Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

Samulski

Chang

Shenk

1989

J Virol

674

280

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A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. transActing AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

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Section: Ap Asmidsmentioning

confidence: 99%

“…Adeno-associated virus (AAV) is a defective parvovirus whose genome is encapsidated as a single-stranded DNA molecule of either plus or minus polarity (3,5,21,23). Although AAV can replicate under appropriate conditions in the absence of a helper virus (29), efficient replication requires coinfection with a helper virus such as adenovirus.…”

mentioning

confidence: 99%

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

Samulski

Chang

Shenk

1989

J Virol

674

280

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“…Adeno-associated virus serotype 2 (AAV2) is a small nonenveloped virus in the parvoviridae family that requires a helper virus, adenovirus or herpesvirus, for efficient productive infection (Casto et al, 1967;McPherson et al, 1985). AAV2 packages a 4681-nucleotide (Srivastava et al, 1983) genome of both positive and negative polarity (Berns and Adler, 1972;Berns and Rose, 1970;Rose et al, 1969) into a 20-to 25-nm virion with icosahedral symmetry (Atchison et al, 1965;Hoggan et al, 1966). The viral DNA consists of two open reading frames flanked by inverted terminal repeats.…”

Section: Introductionmentioning

confidence: 99%

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

1999

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The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.

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“…Studies on the physical and genetic structure of the AAV genome and AAV RNAs have yielded a large amount of inforimation on the replication and organization of the AAV genome. AAV contains a linear, single-stranded genome of 4.7 kilobases (kb) which has been completely sequenced (5,7,34,35,50). The genome is flanked by 145-base terminal repeats (TRs) which are required as origins and primers for DNA replication (21 34; for a review, see reference 6).…”

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confidence: 99%

Positive and negative autoregulation of the adeno-associated virus type 2 genome

Labow¹,

Hermonat²,

Berns³

1986

J Virol

177

134

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The defective human parvovirus, adeno-associated virus (AAV), requires multiple functions provided by a coinfecting helper virus for viral replication. In addition, it has recently been shown that at least one AAV gene is also required for AAV DNA replication. In this paper, we investigate the autoregulation of the AAV genome by analyzing the expression of mutant AAV genomes upon transfection into adenovirus-infected human cells. Evidence is presented which indicates that the AAV genome regulates its own gene expression in at least two ways. First, either the AAV p5 gene or both the p5 and p19 genes appear to encode a trans activator of AAV transcription. Frameshift mutations within the p5 or p19 gene severely inhibited the synthesis and accumulation of all AAV transcripts. The defective accumulation of transcripts could be complemented in trans, in a manner independent of DNA replication, by cotransfection with a capsid deletion mutant. Second, evidence is presented which suggests that the p5 and p19 genes contain negative cis-active regulatory elements. Deletion of sequences within the p5 and p19, genes enhanced the accumulation of the p5 transcript in cis upon complementation with an AAV capsid deletion mutant, whereas certain deletions enhanced p40 RNA accumulation in the absence of trans activation by the p5 gene.

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Separation of Two Types of Adeno-Associated Virus Particles Containing Complementary Polynucleotide Chains

Abstract: An adeno-associated virus containing bromodeoxyuridine-substituted deoxyribonucleic acid has been fractionated by equilibrium centrifugation in CsCl into two classes of virions which contain complementary polynucleotide chains.

Cited by 84 publications

References 7 publications

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

Positive and negative autoregulation of the adeno-associated virus type 2 genome

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