Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

Abstract

ABSTRACT Glycoprotein 3 (GP3) of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region, and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells, GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C terminus of GP3, fused to green fluorescent protein (GFP), is resistant to proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3, and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region forms an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of GP3 and in its hydrophobic face enhances it. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted. IMPORTANCE PRRSV is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here, we analyzed basic structural features of GP3 from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 plays a role during PRRSV infection of pigs: it might serve as a decoy to distract antibodies away from virus particles.