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Adeno-Associated Virus VP1u Exhibits Protease Activity

Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
Author to whom correspondence should be addressed.
Viruses 2019, 11(5), 399;
Received: 27 March 2019 / Revised: 18 April 2019 / Accepted: 19 April 2019 / Published: 29 April 2019
(This article belongs to the Special Issue New Insights into Parvovirus Research)
PDF [2074 KB, uploaded 29 April 2019]


Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical. View Full-Text
Keywords: Adeno-associated virus; AAV; protease; phospholipase-A2; PLA2 Adeno-associated virus; AAV; protease; phospholipase-A2; PLA2

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Kurian, J.J.; Lakshmanan, R.; Chmely, W.M.; Hull, J.A.; Yu, J.C.; Bennett, A.; McKenna, R.; Agbandje-McKenna, M. Adeno-Associated Virus VP1u Exhibits Protease Activity. Viruses 2019, 11, 399.

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