Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology

Abstract

The glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory 1 syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly 2 glycosylated domain, a short hydrophobic region and an unglycosylated C-3 terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus 4 particles, but secretion of GP3 from cells has also been reported. 5We analyzed the membrane topology of GP3 from various PRRSV strains. A 6 fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 7 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion 8 behavior is reversed after exchange of the variable C-terminal domain. A 9 fluorescence protease protection assay shows that the C-terminus of GP3, fused 10 to GFP, is resistant against proteolytic digestion in permeabilized cells. 11Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. 12Both experiments indicate that the C-terminus of GP3 is translocated into the 13 lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic 14 region greatly enhances secretion of GP3 and fusion of this domain to GFP 15 promotes membrane anchorage. Bioinformatics suggests that the hydrophobic 16 region might form an amphipathic helix. Accordingly, exchanging only a few 17 amino acids in its hydrophilic face prevents and in its hydrophobic face enhances 18 secretion of GP3. Exchanging the latter amino acids in the context of the viral 19 genome did not affect release of virions, but released particles were not 20 infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that 21 might explain why a fraction of the protein is secreted. 22including the occurrence of highly pathogenic variants in China ((6), related to 48 PRRSV-2) and Eastern Europe ( (7)), strain Lena, related to PRRSV-1). PRRSV 49 cannot be eliminated from pig farms by vaccination due to the large variability 50 between the existing strains. Especially the glycoproteins show strong antigenic 51 drift and exhibit large variation (up to 50%) in their amino acid sequence (8, 9). 52Arterivirus particles contain a multitude of membrane proteins, the disulphide-53 linked GP5/M dimer and the GP2/3/4 complex, the small and hydrophobic E-54 protein and the ORF5a protein (10). The glycoproteins GP2, GP3 and GP4 as 55 well as the E-protein are essential for replication of both EAV and PRRSV. 56Deleting their genes individually from the viral genome does not prevent budding 57 of virus-like particles from transfected cells, but the particles are not infectious 58 (11, 12). More detailed experiments with EAV showed that if just one of the 59 5 genes encoding GP2, GP3 or Gp4 were deleted the resulting particles do not 60 contain the GP2/3/4 complex and reduced amounts of the E protein (11). It was 61 therefore concluded that the GP2/3/4 complex is essential for virus entry, in the 62 case of PRRSV probably due to its interaction with the key cellular receptor . 64Despite the large amount of sequence information on PRRSV genomes o...