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Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.

Publication ,  Journal Article
Horowitz, ED; Rahman, KS; Bower, BD; Dismuke, DJ; Falvo, MR; Griffith, JD; Harvey, SC; Asokan, A
Published in: J Virol
March 2013

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.

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Published In

J Virol

DOI

EISSN

1098-5514

Publication Date

March 2013

Volume

87

Issue

6

Start / End Page

2994 / 3002

Location

United States

Related Subject Headings

  • Virus Uncoating
  • Virology
  • Osmotic Pressure
  • Microscopy, Electron
  • Microscopy, Atomic Force
  • Hydrogen-Ion Concentration
  • Hot Temperature
  • Fluorometry
  • Dependovirus
  • DNA, Viral
 

Citation

APA
Chicago
ICMJE
MLA
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Horowitz, E. D., Rahman, K. S., Bower, B. D., Dismuke, D. J., Falvo, M. R., Griffith, J. D., … Asokan, A. (2013). Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release. J Virol, 87(6), 2994–3002. https://doi.org/10.1128/JVI.03017-12
Horowitz, Eric D., K Shefaet Rahman, Brian D. Bower, David J. Dismuke, Michael R. Falvo, Jack D. Griffith, Stephen C. Harvey, and Aravind Asokan. “Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.J Virol 87, no. 6 (March 2013): 2994–3002. https://doi.org/10.1128/JVI.03017-12.
Horowitz ED, Rahman KS, Bower BD, Dismuke DJ, Falvo MR, Griffith JD, et al. Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release. J Virol. 2013 Mar;87(6):2994–3002.
Horowitz, Eric D., et al. “Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.J Virol, vol. 87, no. 6, Mar. 2013, pp. 2994–3002. Pubmed, doi:10.1128/JVI.03017-12.
Horowitz ED, Rahman KS, Bower BD, Dismuke DJ, Falvo MR, Griffith JD, Harvey SC, Asokan A. Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release. J Virol. 2013 Mar;87(6):2994–3002.

Published In

J Virol

DOI

EISSN

1098-5514

Publication Date

March 2013

Volume

87

Issue

6

Start / End Page

2994 / 3002

Location

United States

Related Subject Headings

  • Virus Uncoating
  • Virology
  • Osmotic Pressure
  • Microscopy, Electron
  • Microscopy, Atomic Force
  • Hydrogen-Ion Concentration
  • Hot Temperature
  • Fluorometry
  • Dependovirus
  • DNA, Viral