Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear. Here, we present the crystal structures of Npro to 1.25 Å resolution. Structures of pre- and postcleavage intermediates identify three catalytically relevant elements. The trapping of the putative catalytic water reveals its distinct roles as a base, acid, and nucleophile. The presentation of the substrate further explains the enigmatic latency of the protease, ensuring a single in cis cleavage. Additionally, we identified a zinc-free, disulfide-linked conformation of the TRASH motif, an interaction hub of immune factors. The structure opens additional opportunities in utilizing Npro as an autocleaving fusion protein and as a pharmaceutical target.
•Putative catalytic water reveals distinct roles as a base, acid, and nucleophile•The structural mechanism explains a single in cis cleavage•The bimodular architecture reflects proteolytic and immunological functions•The structure provides two orthogonal targets for therapy
The pestivirus protease Npro abolishes host cell antiviral defense. Structures by Zögg et al. reveal residues that act as nucleophiles and as oxyanion pockets. The trapped catalytic water has distinct roles as base, acid, and nucleophile, and the substrate binding mode explains the single in cis cleavage.