Structure and function of Gp3 of porcine reproductive and respiratory syndrome virus

猪繁殖与呼吸综合征病毒Gp3的结构和功能

• 批准号: 427209520
• 负责人: Privatdozent Dr. Michael Veit
• 金额: --
• 依托单位: Institut für Virologie (WE05)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427209520?language=en
• 关键词: Structure; function; Gp3; porcine; reproductive

The porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped negative stranded RNA-virus of the family Arteriviridae, is the most important pathogen in the porcine industry. Its membrane contains two glycoprotein spikes, Gp5/M, which is required for virus budding and a heterotrimeric Gp2/3/4 complex, which has been proposed to play a role during virus entry. However, our information about Gp2/3/4 is mainly derived from studies with the equine arteritis virus, the prototype member of the family. Very little is known about the respective proteins of PRRSV, which also exhibit large amino acid variability. We previously studied basic biochemical features of Gp3 from various PRRSV strains. A fraction of the protein is secreted both from infected and transfected cells, Gp3 from PRRSV-1 strains to a greater extent than Gp3 from PRRSV-2 strains. This secretion behaviour is reversed after exchange of the (between strains) variable C-terminal domain. We also showed that the protein exhibits an unusual hairpin-like membrane topology: both the N- and C-terminus are oriented towards the lumen of the endoplasmic reticulum (ER), membrane anchoring might occur via 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. This rather weak membrane anchoring explains why a fraction of the protein is secreted from cells. With this project we want to test the hypothesis that the hydrophobic region of Gp3 indeed forms an amphiphilic helix, as predicted by bioinformatics. We attempt to determine the structure of this region in the absence and presence of various artificial membranes using CD- and NMR-spectroscopy. Using reverse genetics we will investigate whether virus replication in cell culture requires that a fraction of Gp3 is secreted and whether the amino acid sequence of the hydrophobic region is essential, since it is highly conserved between PRRSV strains although amphiphilic helices can be formed by very different peptides. Next, we want to determine whether soluble and/or membrane-bound Gp3 associates with Gp2/4 and how the complex assembles. Finally, using purified ectodomains of Gp2/3/4 which assemble into disulfide-linked dimers and trimers, we will identify the cysteines that connect the proteins by disulfide-linkages and investigate with affinity chromatography whether Gp2/3/4 binds to CD 163, the essential receptor for cell entry of PRRSV.

猪繁殖与呼吸综合征病毒(PRRSV)是一种囊膜负链RNA病毒，属于动脉病毒科，是养猪业最重要的病原。它的膜含有两个糖蛋白尖峰，Gp5/M是病毒萌发所必需的，以及一个异源三聚体GP2/3/4复合体，它被认为在病毒进入过程中发挥作用。然而，我们关于GP2/3/4的信息主要来自对马动脉炎病毒的研究，马动脉炎病毒是该家族的原型成员。人们对PRRSV各自的蛋白质知之甚少，这些蛋白质也表现出很大的氨基酸变异性。我们先前研究了不同PRRSV毒株的GP3的基本生化特征。一部分蛋白质是从感染细胞和转基因细胞中分泌出来的，PRRSV-1株的GP3比PRRSV-2株的GP3分泌的程度更大。这种分泌行为在(菌株之间)可变的C-末端结构域交换后被逆转。我们还发现，该蛋白呈现出一种不同寻常的发夹样膜拓扑结构：N-端和C-端都朝向内质网(ER)的管腔，膜锚定可能通过两亲性螺旋发生。因此，仅在其亲水表面交换几个氨基酸就可以阻止GP3的分泌，而在其疏水表面则能增强它。这种相当弱的膜锚定解释了为什么一部分蛋白质是从细胞中分泌出来的。通过这个项目，我们想要验证这样一个假设，即正如生物信息学预测的那样，GP3的疏水区确实形成了一个两亲性螺旋。我们试图利用CD-和核磁共振波谱在没有和存在各种人工膜的情况下确定该区域的结构。利用反向遗传学，我们将调查病毒在细胞培养中复制是否需要分泌一部分GP3，以及疏水区的氨基酸序列是否是必需的，因为它在PRRSV株之间高度保守，尽管两亲性螺旋可以由非常不同的多肽形成。接下来，我们想要确定可溶性和/或膜结合的GP3是否与GP2/4相关，以及复合体是如何组装的。最后，利用纯化的GP2/3/4胞外结构域组装成二硫键连接的二聚体和三聚体，我们将鉴定以二硫键连接蛋白的半胱氨酸，并用亲和层析法研究GP2/3/4是否与PRRSV细胞进入细胞的关键受体CD163结合。