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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Publications at this Location » Publication #239177

Title: Porcine Reproductive and Respiratory Syndrome Virus Nsp2 Cysteine Protease Domain Possesses Both Trans- and Cis-cleavage Activities

item HAN, JUN - University Of Minnesota
item RUTHERFORD, MARK - University Of Minnesota
item Faaberg, Kay

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2009
Publication Date: 9/1/2009
Citation: Han, J., Rutherford, M.S., Faaberg, K.S. 2009. Porcine Reproductive and Respiratory Syndrome Virus Nsp2 Cysteine Protease Domain Possesses Both Trans- and Cis-cleavage Activities. Journal of Virology. 83(18):9449-9463.

Interpretive Summary: Porcine reproductive and respiratory syndrome virus is the most costly infectious disease of swine in the United States costing producers in excess of $560 million annually. Although vaccines exist, this pig disease remains very difficult to control and eradicate because the virus constantly changes to avoid protection afforded by vaccines. Moreover, the virus replicates and sheds for several months from pigs. The mechanism this virus uses to replicate and cause disease involves several enzymes the virus makes, one of which is called a replicase. In order for this enzyme to reach its mature functional ability, another group of proteins made by the virus, called proteases, must first process the replicase for it to be become functional. In this study we identified critical portions of a key protease involved in making this replicase functional. Through genetic manipulations of this viral protease, we strategically changed selected individual amino acids and found how these changes altered the ability of this virus to replicate. We found that any changes that altered efficient processing of the protease was lethal to the virus. Results of studies reported here provide additional information to scientists and vaccine companies who seek to rationally design improved vaccines against this virus.

Technical Abstract: he 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 (aa47-180) or the immediate downstream region (aa181-323) was lethal to the virus. In this report, the PL2 domain was found to encode an active enzyme that mediated 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 aa47-240 (Tyr47-Cys240) and aa47-323 (Tyr47-Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55-His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142 and Cys147). The conserved aspartic acids (e.g. Asp85, Asp89) were essential only for PL2 protease trans cleavage activity. Reverse genetics revealed that 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 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide, corresponding to the previously predicted cleavage site.