An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

Abstract

The highly pathogenic porcine reproductive and respiratory virus (hpPRRSV) with discontinuous 30 amino acid (aa) deletion as a gene marker has caused great economic loss in pig industry and since 2007 has become the dominant strain prevalent in China and Vietnam since 2007. Reverse genetics method is a powerful tool urgently needed to be used on the hpPRRSV to study the intriguing molecular mechanism of transcription, replication and the virulence determinant factors. In our study, we successfully constructed a full-length infectious clone, prBJSY07, based on hpPRRSV isolate, BJSY07. The rescued virus, vrBJSY07, showed similar growth characters to those of the parental virus, BJSY07. We also found that a rescued virus vBJSY07 generated from pBJSY07 was viable but displayed decreased and delayed reproductive capacity, which might be caused by two amino acids mutations, S83C and S117C in glycoprotein 3 (GP3), acquired during the preparation of the infectious clone. The topology of the wild type GP3 and the mutant were further analyzed by bioinformatics and revealed that the mutated GP3 possessed slightly altered structure, most likely by forming a new disulfide bond between the two new cysteine residues. As GP3 is a cysteine-rich glycoprotein, common for viral glycoproteins, our results show that GP3 can accommodate even more cysteine mutations. Based on this, a topological model of GP3 is proposed by addressing the intrachain disulfides.

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus

“…ORF3 of PRRSV encodes the GP3 protein, which shows approximately 54% to 60% amino acid identity between North American and European isolates. In addition, GP3 can accommodate cysteine mutations and influence the reproductive capacity of PRRSV [ 9 ]. Experiments using a specific monoclonal antibody against the GP3 protein of the LV strain suggested that the GP3 protein is inserted into the virus particle or is a virus envelope-associated protein [ 10 ].…”

“…GP5 has six epitopes that can induce specific neutralizing antibodies. The neutralizing ability of the antibodies is stronger than those induced by GP4 [ 2 , 9 , 11 ].…”