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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY Title: Proteolytic processing of Porcine Reproductive and Respiratory Syndrome Virus nsp2 replicase protein

Authors
item Han, Jun - UNIVERSITY OF MINNESOTA
item Rutherford, Mark - UNIVERSITY OF MINNESOTA
item Faaberg, Kay

Submitted to: Porcine Reproductive and Respiratory Syndrome International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: October 15, 2007
Publication Date: November 30, 2007
Citation: Han, J., Rutherford, M.S., Faaberg, K.S. 2007. Proteolytic processing of PRRSV nsp2 replicase protein [abstract]. International Porcine Reproductive and Respiratory Syndrome (PRRS) Symposium. Paper No. 23.

Technical Abstract: One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in replication by virus-encoded proteinases. PRRSV nsp2 has been predicted to be the largest viral replicase protein and contains multiple domains: an N-terminal cysteine proteinase (PL2) domain, a middle hypervariable region, and C-terminal putative transmembrane domain. In this study, we investigated the proteolytic processing of nsp2 in PRRSV-infected MARC-145 cells. To facilitate analysis of nsp2 processing, a c-myc epitope was inserted into the nsp2 middle region of a Type 2 strain VR-2332 infectious clone. Recombinant virus was recovered and shown to have a growth rate similar to parental virus. Immunoprecipitation and western blot analysis using a panel of antibodies revealed that several nsp2 associated processing products could be detected. Nsp2 proteins corresponding to sizes with and without the predicted transmembrane domain were observed. A pulse-chase assay suggested that these nsp2 products appeared rapidly, possessed an extended life-span and a low turnover rate. Further deletion mutants were used to map the relative positions of the cleavage sites. Finally, mass spectrometry and other biochemical approaches were used to locate the nsp2 cleavage sites. The finding of different PRRSV nsp2 isoforms may suggest that they have different roles in the virus life cycle.

Last Modified: 12/21/2014
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