Adeno-Associated Virus VP1u Exhibits Protease Activity

• Main Authors: Justin J. Kurian, Renuk Lakshmanan, William M. Chmely, Joshua A. Hull, Jennifer C. Yu, Antonette Bennett, Robert McKenna, Mavis Agbandje-McKenna
• Format: Article
• Language: English
• Published: MDPI AG 2019-04-01
• Series: Viruses
• Subjects: Adeno-associated virus; AAV; protease; phospholipase-A₂; PLA₂
• Online Access: https://www.mdpi.com/1999-4915/11/5/399

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.