Signal peptide cleavage from GP3 enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic C-terminus is essential

Matczuk, Anna Karolina; Veit, Michael

doi:10.1016/j.virusres.2014.02.005

Cited by 10 publications

(10 citation statements)

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“…This indicates that co-translational attachment of carbohydrates prevents access of the signal peptidase to the cleavage site of Gp3 (Matczuk et al, 2013). An identical result was obtained when the overlapping sequon was exchanged in the context of the viral genome (Matczuk and Veit, 2014). Since glycosylation usually does not occur close to membranespanning segments (Nilsson and von Heijne, 1993), we analysed the topology of Gp3.…”

Section: Gp3mentioning

confidence: 62%

“…When the signal peptide of Gp3 is cleaved (upon mutation of the overlapping sequon), the infectivity of the recombinant EAV is not impaired, and Gp3 with cleaved signal peptide associates with Gp2/4 in virus particles. In contrast, viruses containing Gp3 with deleted hydrophobic C-terminus rapidly reverted back to wild type (Matczuk and Veit, 2014).…”

Section: Gp3mentioning

confidence: 96%

“…Furthermore, mutations in the N-terminal region of Gp3 occurring during virus propagation in animals might have a similar effect. We have shown that EAV containing Gp3 with cleaved signal peptide is as infectious as wild type EAV, at least in cell culture (Matczuk and Veit, 2014). EAV isolated from the semen of a persistently infected stallion has lost the glycosylation site in the N-terminal region of Gp3, which is otherwise highly conserved .…”

Section: Creating Protein Diversity By Competition Between Co-translamentioning

confidence: 96%

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Membrane proteins of arterivirus particles: Structure, topology, processing and function

et al. 2014

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Arteriviruses, such as equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), are important pathogens in veterinary medicine. Despite their limited genome size, arterivirus particles contain a multitude of membrane proteins, the Gp5/M and the Gp2/3/4 complex, the small and hydrophobic E protein and the ORF5a protein. Their function during virus entry and budding is understood only incompletely. We summarize current knowledge of their primary structure, membrane topology, (co-translational) processing and intracellular targeting to membranes of the exocytic pathway, which are the budding site. We profoundly describe experimental data that led to widely believed conceptions about the function of these proteins and also report new results about processing steps for each glycoprotein. Further, we depict the location and characteristics of epitopes in the membrane proteins since the late appearance of neutralizing antibodies may lead to persistence, a characteristic hallmark of arterivirus infection. Some molecular features of the arteriviral proteins are rare or even unique from a cell biological point of view, particularly the prevention of signal peptide cleavage by co-translational glycosylation, discovered in EAV-Gp3, and the efficient use of overlapping sequons for glycosylation. This article reviews the molecular mechanisms of these cellular processes. Based on this, we present hypotheses on the structure and variability of arteriviral membrane proteins and their role during virus entry and budding.

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Section: Gp3mentioning

confidence: 62%

Section: Gp3mentioning

confidence: 96%

Section: Creating Protein Diversity By Competition Between Co-translamentioning

confidence: 96%

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Membrane proteins of arterivirus particles: Structure, topology, processing and function

et al. 2014

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“…This region (RPTLICWFALLLVHFLPMPRCRGS) exhibits no sequence homology to the presumed helices of PRRSV and LDV, but heliquest predicts that it forms an amphipathic helix with similar biophysical properties (<μH>: 0.258; <H>: 1.181 for underlined amino acids) than the helices of PRRSV and LDV. Similar to PRRSV,membrane-anchoring of GP3 of EAV is also essential for virus replication since infectious mutants with a stop codon inserted into this region could not be generated (34). In sum, although experimentally not analyzed for each protein, GP3 proteins from PRRSV, LDV and EAV might have the same hairpin-like membrane topology and the same mechanism of membrane-anchoring, but the presumed amphipathic helix is formed by different amino acids in the genera Rodartevirus (LDV and PRRSV) and Equartevirus (EAV).…”

Section: Discussionmentioning

confidence: 99%

“…However, the uncleaved signal peptide does not act as a membrane anchor. Membrane attachment is caused by the hydrophobic C-terminus of GP3, which does not span the membrane, but rather attaches the protein peripherally to ER membranes (33, 34). In contrast, the signal peptide of GP3 from PRRSV (and of LDV) is cleaved despite the presence of a carbohydrate in its vicinity (35) indicating that there are differences in protein processing between the Arterivirus genera Rodartevirus (LDV and PRRSV) and Equartevirus (EAV).…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Krabben

Wang

et al. 2018

Preprint

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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...

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Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins

Zhang,

Qian,

Veit

2024

Veterinary Microbiology

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Signal peptide cleavage from GP3 enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic C-terminus is essential

Cited by 10 publications

References 28 publications

Membrane proteins of arterivirus particles: Structure, topology, processing and function

Membrane proteins of arterivirus particles: Structure, topology, processing and function

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

Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins

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