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

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