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

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. 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.

Charged Residues in the Membrane Anchor of the Pestiviral Erns Protein Are Important for Processing and Secretion of Erns and Recovery of Infectious Viruses

“…This helix associates in plane with the lipid bilayer surface [ 25 , 26 , 27 ]. Among surface proteins, this type of membrane anchor is only found in E rns and the GP3 envelope protein of PRRSV [ 29 ]. Association to membranes via the amphipathic helix is believed to be responsible for partial secretion of both of these proteins, ensuring an equilibrium between secretion and retention.…”

“…A further unusual feature of this protein is its unusual membrane anchor in the form of a long carboxy-terminal amphipathic helix that aligns in plane with the membrane surface [ 25 , 26 , 27 , 28 ]. This kind of membrane association is unknown for cellular surface proteins and has only been reported for one other viral envelope protein so far, the glycoprotein 3 (GP3) of porcine respiratory, and reproductive syndrome virus (PRRSV) [ 29 ]. Both E rns and GP3 are secreted to significant amounts by the infected cells [ 10 , 25 , 26 , 27 , 29 ], which, for the pestivirus protein, is believed to be connected with its inhibitory effect on the innate immune system.…”

“…This kind of membrane association is unknown for cellular surface proteins and has only been reported for one other viral envelope protein so far, the glycoprotein 3 (GP3) of porcine respiratory, and reproductive syndrome virus (PRRSV) [ 29 ]. Both E rns and GP3 are secreted to significant amounts by the infected cells [ 10 , 25 , 26 , 27 , 29 ], which, for the pestivirus protein, is believed to be connected with its inhibitory effect on the innate immune system. In infected animals, considerable amounts of E rns are found in the blood [ 19 ].…”

Glycoprotein 5 Is Cleaved by Cathepsin E during Porcine Reproductive and Respiratory Syndrome Virus Membrane Fusion

“…Previous evidence shows that GP4 protein is a glycosyl-phosphatidylinositol (GPI)-modified membrane-associated protein with two predicted hydrophobic domains: an N-terminal domain as the signal peptide and a C-terminal domain to anchor the protein to membrane (54). A recent work reports that PRRSV GP3 consists of a cleaved signal peptide, a highly glycosylated domain, an unglycosylated C-terminal domain, and a hydrophobic region exhibiting an unusual hairpin-like membrane topology (amphiphilic helix) (55). Although these proteins do not comply with the properties of typical viral fusion proteins, we cannot preclude their involvement in PRRSV membrane fusion, which needs to be clarified in the future.…”

Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP3 Regulates Claudin 4 To Facilitate the Early Stages of Infection

“…The PRRSV structural protein genes of GP2, GP3, GP4, GP5, and M (GenBank accession number HQ416720.1), CLDN4 (GenBank accession number NM_001194564), and the ECL1 and ELC2 loops of CLDN4 were amplified by reverse transcription PCR (RT-PCR) or annealed and cloned into the pEGFP-C1 vector (Clontech Laboratories, Inc., Mountain View, CA, USA) together with the PRRSV GP2, GP3, GP4, GP5, and M genes. GP3 contains a signal peptide (SP), N-terminal domain, hydrophobic region, and C-terminal domain (62). The names and sizes of truncated GP3 are shown in Fig.…”