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

猪繁殖与呼吸综合征病毒（PRRSV）是猪动脉病毒科的一种包膜负链rna病毒，是养猪业中最重要的病原体。它的膜包含两个糖蛋白尖刺，Gp5/M，这是病毒出芽所必需的，还有一个异源三聚体Gp2/3/4复合物，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（该家族的原型成员）的研究。我们对PRRSV各自的蛋白质知之甚少，它们也表现出很大的氨基酸变异性。我们之前研究了不同PRRSV毒株Gp3的基本生化特征。PRRSV-1株的Gp3蛋白比PRRSV-2株的Gp3蛋白分泌量更大。在菌株之间交换可变c端结构域后，这种分泌行为被逆转。我们还发现该蛋白呈现出一种不寻常的发夹状膜拓扑结构：N端和c端都指向内质网（ER）的管腔，膜锚定可能通过两向螺旋发生。因此，在其亲水性面交换少量氨基酸可以阻止Gp3的分泌，而在其疏水性面交换则可以促进Gp3的分泌。这种相当弱的膜锚定解释了为什么一部分蛋白质会从细胞中分泌出来。在这个项目中，我们想要验证Gp3的疏水区域确实形成了一个两亲性螺旋的假设，正如生物信息学预测的那样。我们试图确定该区域的结构，在缺席和存在的各种人工膜使用CD-和核磁共振光谱。利用反向遗传学，我们将研究病毒在细胞培养中的复制是否需要分泌Gp3的一部分，以及疏水区的氨基酸序列是否必要，因为它在PRRSV菌株之间是高度保守的，尽管两亲螺旋可以由非常不同的肽形成。接下来，我们想要确定可溶性和/或膜结合的Gp3是否与Gp2/4结合，以及该复合物如何组装。最后，利用纯化的Gp2/3/4外结构域组装成二硫键连接的二聚体和三聚体，我们将鉴定通过二硫键连接蛋白质的半胱氨酸，并通过亲和层析分析Gp2/3/4是否与PRRSV进入细胞的必需受体cd163结合。

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.