Processing and Function of the Gp2/3/4 Spike of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

动脉病毒科是一个囊膜RNA病毒家族，尽管它们在兽医中很重要，但只有很差的特征。马动脉炎病毒(Eav)是该家族的原型成员，会引起马的大量疾病，而猪繁殖与呼吸综合征病毒(PRRSV)是养猪业最重要的病原体。PRRSV引起的持续性感染，除了其糖蛋白的高度变异性外，也是从养猪场消除病毒的主要障碍。持续存在的一个可能的分子原因是中和抗体出现得较晚，但它们的蛋白质靶标和表位尚未确定。动脉病毒的包膜含有两个糖蛋白尖峰，Gp5/M和GP2/3/4，它们位于内质网或早期高尔基体细胞中，内质网或早期高尔基体是病毒颗粒的组装部位。反向遗传实验表明，萌发需要Gp5/M，细胞进入需要Gp2/3/4。GP2/3/4被认为是将动脉病毒附着在细胞受体上，并催化膜融合。二硫化物连接的GP2/3/4尖峰是通过一个复杂的、基本上不被理解的过程组装的。而GP2和GP4在细胞内形成二硫键连接的二聚体，推测是在内质网中，而GP3和GP2/4之间的二硫键只在萌芽的病毒颗粒中形成。我们最近对EAV的研究揭示了GP3的另一个独特特征：位于信号肽附近的N-连接碳水化合物抑制其切割。这些研究还导致了一种新的GP3膜拓扑结构模型：未切割的信号肽不作为膜锚，而是完全转运到内质网的管腔中。锚定是由疏水的C-末端引起的，它不是跨膜区，而是将GP3外围连接到膜上。通过我们的研究项目，我们想要解开GP2/3/4的组装途径，并创建一个工具来分析它的功能。我们将首先分析不同PRRSV毒株的GP3是否遵循相同的独特加工方案，并显示出与EAV株GP3相同的膜拓扑结构。有趣的是，PRRSV毒株在GP3 C末端的生物物理性质上不同，这表明在某些毒株中，GP3可能不是膜结合的，而是由细胞分泌的。反向遗传学将被用来分析GP3膜锚定对病毒感染性的意义。然后，我们将在转基因细胞中从其成分中组装出天然的GP2/3/4复合体。保留信号的去除将允许复合体靶向质膜，保留信号尚未被鉴定，但很可能位于蛋白质的跨膜区。暴露在表面的GP2/3/4复合体适用于多种检测方法，如与细胞受体的结合、与抗体的结合和膜融合试验，因此有助于阐明GP2/3/4刺激物在病毒入侵过程中的作用，以及作为中和抗体的假定靶标。

Arteriviridae are a family of enveloped RNA viruses, which are, despite their importance in veterinary medicine, only poorly characterized. The equine arteritis virus (EAV) is the prototype member of the family and causes substantial disease in horses, whereas the porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the porcine industry. PRRSV causes persistent infection, which is, besides the high variability of its glycoproteins, the main obstacle to eliminate the virus from pig farms. One possible molecular cause of persistence is the late appearance of neutralizing antibodies, but their protein targets and epitopes have not been identified. The envelope of arteriviruses contain two glycoprotein spikes, Gp5/M and Gp2/3/4, which are in cells retained in the ER or early-Golgi, the assembly site of virus particles. Reverse genetic experiments indicate that Gp5/M is required for budding and Gp2/3/4 for cell entry. Gp2/3/4 is supposed to attach arteriviruses to cellular receptors and to catalyse membrane fusion. The disulphide-linked Gp2/3/4 spike is assembled by a complicated and largely uncomprehended process. Whereas Gp2 and Gp4 form a disulphide-linked dimer inside cells, presumably in the ER, disulphide-linkages between Gp3 and Gp2/4 only form in budded virus particles. Our recent work with EAV revealed another unique feature of Gp3: N-linked carbohydrates located adjacent to the signal peptide inhibit its cleavage. These studies also led to a new model for the membrane topology of Gp3: The uncleaved signal peptide does not act as a membrane anchor but is completely translocated into the lumen of the ER. Anchoring is caused by the hydrophobic C-terminus, which is not a transmembrane region, but attaches Gp3 peripherally to membranes. With our research project we want to unravel the assembly pathway of Gp2/3/4 and create a tool to analyse its function. We shall first analyse whether Gp3 from various PRRSV strains follows the same unique processing scheme and exhibits the same membrane topology as Gp3 from EAV. Interestingly, PRRSV strains differ in the biophysical properties of the C-terminus of Gp3 suggesting that in some strains Gp3 might not be membrane bound, but secreted from cells. Reverse genetics will be used to analyse the significance of membrane anchoring of Gp3 on the infectivity of viruses. We shall then assemble a native Gp2/3/4 complex in transfected cells from its components. Removal of retention signals, which have not been identified but are likely to be located in the transmembrane region of the proteins, will allow targeting of the complex to the plasma membrane. A surface exposed Gp2/3/4 complex is amenable to a multitude of examination methods, such as attachment to cellular receptors, binding to antibodies and membrane fusion assays and is thus instrumental to elucidate the role of the Gp2/3/4 spike during virus entry and as a putative target for neutralizing antibodies.