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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY Title: Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture

Authors
item Han, Jun - UNIVERSITY OF MINNESOTA
item Liu, Gongping - UNIVERSITY OF MINNESOTA
item Wang, Yue - UNIVERSITY OF MINNESOTA
item Faaberg, Kay

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 16, 2007
Publication Date: September 1, 2007
Citation: Han, J., Liu, G., Wang, Y., Faaberg, K.S. 2007. Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture. Journal of Virology. 81(18):9878-9890.

Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) causes a swine disease that is of great economic concern. Present vaccines against PRRSV are not ideal. In order to develop a better vaccine, we developed a system to allow changes to the virus that we then can analyze. We are now assessing the need for different regions of the viral make-up, one being a region known as nonstructural protein 2, which is part of the machinery that replicates the virus. In this publication, we determined that much of this protein is not necessary for viral growth in cells, most of the recovered viruses had little defect in growth compared to the wild-type virus and that a gene marker inserted into the virus was not stable. The results will be used by scientists to produce vaccine constructs and to increase their knowledge of why PRRSV causes disease. The downstrean impact on swine producers, industry and the general public will be a better and safer vaccine for less cost.

Technical Abstract: The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is a multi-domain protein and has been shown to undergo remarkable genetic variation, primarily in its middle region, while exhibiting high conservation in the N-terminal putative protease domain and the C-terminal predicted transmembrane region. A reverse genetic system of PRRSV North American prototype VR-2332 was developed to explore the importance of different regions of nsp2 for viral replication. A series of mutants with in-frame deletions in the nsp2 coding region were engineered and infectious viruses were subsequently recovered from transfected cells and further characterized. The results demonstrated that the cysteine protease domain (PL2), the PL2 downstream flanking sequence (aa181-323) and the putative transmembrane domain were critical for replication. By contrast, the segment of nsp2 preceding the PL2 domain (aa13-35) was dispensable for viral replication, and the nsp2 middle hypervariable region (aa324-813) tolerated 100aa or 200aa deletions but could not be removed as a whole; the largest deletion was about 400aa (nsp2delta324-726). Characterization of the mutants demonstrated that those with small deletions possessed growth kinetics and RNA expression profiles similar to the parental virus, while the nsp2delta324-726 mutant displayed decreased cytolytic activity on MARC-145 cells and did not develop visible plaques. Finally, the utilization of the genetic flexibility of nsp2 to express foreign genes was examined by inserting the gene encoding green fluorescent protein (GFP), in frame, into one nsp2 deletion mutant construct. The recombinant virus was viable but impaired and unstable, and gradually gained parental growth kinetics by the loss of most of the GFP gene.

Last Modified: 9/2/2014
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