A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine... Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M r ) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP 3 ) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP 3 protein was present in amounts equivalent to those of the N, M, and GP 5 proteins in the infected cells, whereas no GP 3 could be detected in purified virions. During the first 30 min of chase, the GP 3 undergoes a gradual downward shift of its apparent M r , thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

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Publisher
Springer-Verlag
Copyright
Copyright © Wien by 1998 Springer-Verlag/
Subject
Legacy
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s007050050430
Publisher site
See Article on Publisher Site

Abstract

Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M r ) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP 3 ) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP 3 protein was present in amounts equivalent to those of the N, M, and GP 5 proteins in the infected cells, whereas no GP 3 could be detected in purified virions. During the first 30 min of chase, the GP 3 undergoes a gradual downward shift of its apparent M r , thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural.

Journal

Archives of VirologySpringer Journals

Published: Oct 1, 1998

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