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Cited by 136Virus-based gene therapy researchRNA Interference and Gene DeliveryViral Infectious Diseases and Gene Expression in Insects

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

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Rabinowitz, Joseph E., Xiao, Weidong, Samulski, R.Jude·1999·Virology
DOI 10.1006/viro.1999.0045
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Abstract

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 descri…

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Crossref · 10.1006/viro.1999.0045

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  • Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2.

    “Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2 [31–34].”

    From Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses (2017) · cites this paper as [33]

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Authors
Rabinowitz, J. E., Xiao, W., & Samulski, R. J.
Affiliations
  • University of North Carolina at Chapel Hill (US)
  • University of Pennsylvania (US)
ORCID
Venue
Virology
Publisher
Elsevier BV
Published
1999-12
Volume
265
Issue
2
Pages
274-285
ISSN
0042-6822 (ISSN-L)
DOI
10.1006/viro.1999.0045
OpenAlex
W2025522522
Cited by
136
References
1
Citations by year
2026: 1 · 2025: 1 · 2024: 4 · 2023: 2 · 2022: 4 · 2021: 1 · 2020: 1 · 2019: 6 · 2018: 2 · 2017: 3 · 2016: 1 · 2015: 4 · 2014: 3 · 2013: 1 · 2012: 4
FWCI
4.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Virology (publishedVersion)
  • PubMed (publishedVersion)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
  • UNC Libraries
Funders
  • Muscular Dystrophy Association
  • National Institutes of Health (1r21dk053423-01)
  • National Heart, Lung, and Blood Institute (HL51818-06, P01HL051818)
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK53423-01)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords
AAV2, capsid, insertional mutagenesis, viral assembly, viral infectivity, heparin binding, VP3, capsid mutants, recombinant AAV production, gene delivery vectors
MeSH terms
Dependovirus, Capsid, Cell Line, Transformed, HeLa Cells, Heparin, Humans, Recombination, Genetic, Virion, Virus Replication, Mutagenesis, Insertional, Capsid Proteins

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