Allosteric regulation of Senecavirus A 3C(pro) proteolytic activity by an endogenous phospholipid

Author summarySeneca virus A (SVA) is a novel picornavirus and there have been several recent outbreaks that caused a great threat to the swine industry in some countries. The protease 3C(pro) is responsible for the cleavage of viral polyprotein and it can also cleave several host proteins enabling SVA to escape antiviral innate immune responses. We identified an endogenous phospholipid molecule that binds to a unique region in SVA 3C(pro) by a combination of crystallography, untargeted lipidomics, and immunoblotting. The preferred phospholipid types include cardiolipin, phosphoinositol-4-phosphate and sulfatide. We found the protease activity can be significantly inhibited when the phospholipid-binding capacity decreases. Meanwhile, a decrease in infectivity titers of SVA mutants harboring mutations that impair the lipid-binding ability of 3C(pro) was observed. Our findings suggest the phospholipid may function as an allosteric activator to regulate SVA 3C(pro) proteolytic activity associated with the viral replication and infection. Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3C(pro)) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3C(pro) with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3C(pro). Our lipid-binding assays showed that SVA 3C(pro) displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3C(pro) was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3C(pro)-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3C(pro) structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3C(pro), indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3C(pro), suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme's proteolytic activity during infection.