Affiliation:
1. Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108
2. Virus and Prion Diseases of Livestock, National Animal Disease Center, USDA-Agricultural Research Service, 2300 Dayton Avenue, Ames, Iowa 50010
Abstract
ABSTRACT
The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both
trans
- and
cis
-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr
47
to Cys
240
) and aa 47 to 323 (Tyr
47
to Leu
323
), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys
55
- His
124
catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys
111
, Cys
142
, and Cys
147
). The conserved aspartic acids (e.g., Asp
89
) were essential for the PL2 protease
trans
-cleavage activity. Reverse genetics revealed that the PL2
trans
-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease
trans
function, but not the
cis
activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G
1196
|G
1197
dipeptide.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology