N-Terminal Protease of Pestiviruses: Identification of Putative Catalytic Residues by Site-Directed Mutagenesis

Pestiviruses are the only members of the Flaviviridae that encode a nonstructural protease at the N terminus of their polyproteins. This N-terminal protease (N(pro)) cleaves itself off of the nascent polyprotein autocatalytically and thereby generates the N terminus of the adjacent viral capsid protein C. In previous reports, sequence similarities between N(pro) and the catalytic residues of papain-like cysteine proteases were put forward. To test this hypothesis, substitutions of cysteine and histidine residues within N(pro) were carried out by site-directed mutagenesis. Translation of the mutagenized N(pro)-C proteins in cell-free lysates confirmed that only the predicted Cys(69) was an essential amino acid for proteolysis, not His(130). Further essential residues were identified with His(49) and Glu(22). While it remains speculative whether Glu(22)-His(49)-Cys(69) actually build a catalytic triad, these results invalidate the assumption that N(pro) is a papain-like cysteine protease.