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FEBS J. 2019 Jul 22. doi: 10.1111/febs.15013. [Epub ahead of print]

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

Author information

1
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India.
2
SASTRA University, Thanjavur, India.
3
Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, NE, USA.
4
Department of Haematology and Centre for Stem Cell Research, Vellore, India.

Abstract

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 3.4.21.4.

KEYWORDS:

AAV ; LCMS ; MALDI-TOF analysis; capsid; post-translational modification

PMID:
31330090
DOI:
10.1111/febs.15013

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