A NONSTRUCTURAL AND ANTIGENIC GLYCOPROTEIN IS ENCODED BY ORF3 OF THE IAF-KLOP STRAIN OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was revers e-transcribed and cloned into the procaryotic expression vector pGEX-4 T-1, then subcloned into the eucaryotic expression vector pAdCMV5 whic h was used as a shuttle vector to generate a replication-defective rec ombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion prote in was used to raise a monospecific antiserum in rabbits. By Western-i mmunoblotting with PRRSV-infected cell extracts, the ORF3 encoded prot ein had an estimated molecular mass (M-r) of 42 kDa, similar to that o f the protein expressed by the adenovirus vector. Endoglycosidase F di gestion showed that the ORF3 encoded protein occurs in an highly glyco sylated form (GP(3)) in the infected MARC-145 cells. Pulse-chase and r adioimmunoprecipitation experiments revealed that the GP(3) protein wa s present in amounts equivalent to those of the N, M, and GP(5) protei ns in the infected cells, whereas no GP3 could be detected in purified virions. During the first 30 min of chase, the GP(3) undergoes a grad ual downward shift of its apparent M-r, thought to result from trimmin g of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluoresc ence reacted positively with the recombinant GST-ORF3 fusion protein b y immunoblotting. Data indicated that the ORF3 protein of the Quebec r eference strain of PRRSV is a highly glycosylated and antigenic protei n, which is nonstructural.