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.