Adeno-Associated Virus VP1u Exhibits Protease Activity

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Adeno-associated viruses (AAVs) are crucial for gene therapy, with numerous clinical trials targeting monogenic diseases.
• The unique N-terminus of AAV VP1 (VP1u) contains a phospholipase A2 (PLA2) enzyme domain.

Purpose of the Study:

• To investigate the functional properties of the AAV VP1u domain beyond its canonical PLA2 activity.
• To determine if VP1u possesses other enzymatic functions, specifically protease activity.

Main Methods:

• Assessing VP1u's proteolytic activity against various protein substrates (casein, gelatin, BSA).
• Evaluating the effect of inhibitors (EDTA, EGTA, A2M) on VP1u protease activity.
• Performing site-directed mutagenesis of the VP1u PLA2 catalytic motif and other residues to probe the relationship between PLA2 and protease functions.

Main Results:

• AAV VP1u demonstrated protease activity against casein and gelatin, but not native BSA.
• Heated BSA, a disordered protein, was susceptible to VP1u-mediated cleavage.
• Protease activity was partially inhibited by chelators and A2M, and mutations in the PLA2 active site or at H38 affected this activity, indicating a complex relationship.

Conclusions:

• The AAV VP1u domain possesses a distinct protease function that targets disordered proteins.
• This protease activity is associated with, yet mechanistically distinct from, its canonical PLA2 function.
• Understanding VP1u's dual enzymatic nature may offer new insights into AAV biology and gene delivery applications.