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J Virol, March 1998, p. 2544-2547, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Tillmann Rümenapf,* Robert Stark, Manuela Heimann, and Heinz-Jürgen Thiel

Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität, D-35392 Giessen, Germany

Received 20 August 1997/Accepted 24 November 1997

Pestiviruses are the only members of the Flaviviridae that encode a nonstructural protease at the N terminus of their polyproteins. This N-terminal protease (Npro) 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 Npro and the catalytic residues of papain-like cysteine proteases were put forward. To test this hypothesis, substitutions of cysteine and histidine residues within Npro were carried out by site-directed mutagenesis. Translation of the mutagenized Npro-C proteins in cell-free lysates confirmed that only the predicted Cys69 was an essential amino acid for proteolysis, not His130. Further essential residues were identified with His49 and Glu22. While it remains speculative whether Glu22-His49-Cys69 actually build a catalytic triad, these results invalidate the assumption that Npro is a papain-like cysteine protease.

* Corresponding author. Mailing address: Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität, Frankfurter Strasse 107, D-35392 Giessen, Germany. Phone: 49-(641)-99 38351. Fax: 49-(641)-99 38359. E-mail: Till.H.Ruemenapf{at}vetmed.uni-giessen.de.




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