GP3 is a structural component of the PRRSV type II (US) virion

Abstract

Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen to generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, (35)S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.

High Genetic Diversity and Adaptive Potential of Two Simian Hemorrhagic Fever Viruses in a Wild Primate Population

“…GP3 is also immunogenic [44], [45] and glycans attached to the GP3 ectodomain may play a role in evasion of the humoral immune response through the shielding neutralizing antibody epitopes [46]. It is possible that GP3 has multiple functions, as GP3 of PRRSV and LDV have been found in both virion-associated and soluble secreted forms [47]–[51]. Our analysis revealed a distinct region of non-synonymous diversity suggestive of positive selection in ORF3 of SHFV-krc2 (codons 141–173) (Figure 7D).…”

Immune Evasion of Porcine Reproductive and Respiratory Syndrome Virus through Glycan Shielding Involves both Glycoprotein 5 as Well as Glycoprotein 3

“…Anti-GP5 polyclonal antibody was kindly provided by Carl Gagnon (University of Quebec, Montreal, Canada). Anti-GP3 polyclonal antibody was previously described (14). Swine hyperimmunized antisera against PRRSV strains NSVL 97-7895, VR2332, MN184, and Lelystad were prepared as previously described (33).…”

Attenuation and Immunogenicity of a Live High Pathogenic PRRSV Vaccine Candidate with a 32-Amino Acid Deletion in the nsp2 Protein

“…Another residue change (L 48   →  F 48 ) located in ORF2 was also found in two commercial MLV vaccines JXA1R and HUN4-F112, which seemed to be a unique molecular signature among the HP-PRRSV attenuated strains. It was shown that GP3, as a structural protein [43] presenting on virion envelope, was potentially associated with viral attenuation [30]. In this study, mutations (aa, H 79   →  Y 79 , F 143   →  L 143 ) located in the antigenic region of GP3 were detected, and this mutation could influence the characteristics of antigenic sites of GP3.…”