SUMOylation targets AAV capsids but mainly restricts transduction by cellular mechanisms.

Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy, due to its non-pathogenic nature, ease of production and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the Small Ubiquitin-like Modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated as indicated by deletion and point mutations of VP2 or the obstruction of its N-terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability compared to non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation, however, it reduced activation of gene transduction by shut down of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor, that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the co-expression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.ImportanceHost factors within the cell are the major mode of restriction of Adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide siRNA screen, we identified that proteins of the Small Ubiquitin-like Modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV virus directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.