Phosphorylation of enteroviral 2A^pro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis

Enteroviral 2A proteinase (2A^pro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2A^pro ; however, the mechanism underlying the regulation of 2A^pro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2A^pro or Thr125 of coxsackievirus B3 2A^pro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2A^pro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2A^pro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2A^pro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2A^pro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2A^pro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.