An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like...

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CL^pro) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CL^pro exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CL^pro remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CL^pro using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CL^pro numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CL^pro and EAV 3CL^pro formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CL^pro and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CL^pro toward P1-Gln. In turn, the T139K exchange of EAV 3CL^pro showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CL^pro to trigger a substrate specificity switch.