The Arterivirus Nsp4 Protease Is the Prototype of a Novel Group of Chymotrypsin-like Enzymes, the 3C-like Serine Proteases

Eric J Snijder,Alexander E Gorbalenya,Alfred L.M Wassenaar,Willy J.M Spaan,Leonie C Van Dinten

doi:10.1074/jbc.271.9.4864

Abstract

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.

Highlights

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases
Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups
Identification of the Proteolytic Activity and Catalytic Triad of the Equine arteritis virus (EAV) SP—To test the proteolytic function and predicted active site residues for the EAV Nsp4 SP and to establish its role in ORF1a protein processing, the Nsp4 domain was subjected to site-directed mutagenesis

Summary

Introduction

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/ Glu)2(Gly/Ala/Ser) cleavage sites. In a large number of viral CHL proteases, the picornavirus 3C protease being the prototype, Cys replaces Ser as the catalytic nucleophile In addition to this substitution, a number of these “3C-like” enzymes utilize Glu instead of the active site Asp (5, 9 –11). The sites cleaved by 3C-like Cys proteases, for example, are generally similar They usually contain Gln or Glu at the P1 position and a small amino acid residue (Gly, Ala, or Ser) at the P1Ј position [3, 12]. These residues were later shown to be conserved in the sequences of two other arteriviruses, porcine reproductive and respiratory syndrome virus [15] and lactate dehydrogenase-elevating virus [16]

Methods

Results

Conclusion