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

Abstract

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.