Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology
Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology
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DOI：
10.1128/jvi.00660-18
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发表时间：
2018-04
影响因子：
5.4
通讯作者：
Minze Zhang;L. Krabben;Fangkun Wang;M. Veit
中科院分区：
文献类型：
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作者：
Minze Zhang;L. Krabben;Fangkun Wang;M. Veit
猪繁殖与呼吸综合征病毒（PRRSV）的糖蛋白3 （GP3）由一个断裂的信号肽、一个高度糖基化的结构域、一个短的疏水区和一个未糖基化的c端结构域组成。据推测，GP3在病毒颗粒中与GP2和GP4形成复合物，但也有报道称GP3从细胞中分泌出来。我们分析了不同PRRSV菌株GP3的膜拓扑结构。部分蛋白从转染的细胞分泌，PRRSV-1株的GP3比PRRSV-2株的GP3分泌更多。在交换可变c端结构域后，这种分泌行为被逆转。荧光蛋白酶保护实验表明，GP3的C端与绿色荧光蛋白（GFP）融合，在渗透细胞中抵抗蛋白水解消化。此外，还使用了插入GP3 c端部分的糖基化位点。两项实验均表明GP3的C端转运到内质网管腔内。保守疏水区域的缺失极大地促进了GP3的分泌，该区域与GFP的融合促进了膜的锚定。生物信息学表明疏水区形成两亲螺旋结构。因此，在其亲水性面交换少量氨基酸可以阻止GP3的分泌，而在其疏水性面则可以促进GP3的分泌。在病毒基因组的背景下交换后一种氨基酸并不影响病毒粒子的释放，但释放的颗粒不具有传染性。总之，GP3表现出一种不寻常的发夹状膜拓扑结构，这可能解释了为什么会分泌一部分蛋白质。PRRSV是猪肉工业中最重要的病原体。它引起持续性感染，导致仔猪体重增加减少；高致病性菌株甚至杀死90%的受感染猪群。PRRSV不能通过疫苗接种从猪场中消除，因为现有菌株之间的氨基酸差异很大，尤其是糖蛋白。本文分析了不同PRRSV毒株GP3的基本结构特征。我们发现这种蛋白质呈现出一种不寻常的发夹状膜拓扑结构；膜锚定可能通过两性螺旋发生。这种相当弱的膜锚解释了为什么一部分蛋白质会从细胞中分泌出来。有趣的是，PRRSV-1株比PRRSV-2株分泌更多的GP3。我们推测，分泌的GP3在猪PRRSV感染过程中发挥了作用：它可能作为一个诱饵，将抗体从病毒颗粒上转移开。
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.