Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes

FEBS J. 2019 Dec;286(24):4964-4981. doi: 10.1111/febs.15013. Epub 2019 Aug 1.


Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMOylation (13%) and acetylation (11%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had >7 post-translational modifications in their capsid protein. About 82% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~ 100-fold vs. AAV2 wild-type vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. ENZYMES: Trypsin, EC

Keywords: AAV; LCMS; MALDI-TOF analysis; capsid; post-translational modification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Dependovirus
  • Genetic Vectors / genetics
  • Glycosylation
  • HeLa Cells
  • Humans
  • Mutation / genetics
  • Parvovirinae / genetics*
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Serogroup
  • Transduction, Genetic
  • Ubiquitination


  • Capsid Proteins

Supplementary concepts

  • Adeno-associated virus-1