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

动脉病毒科是一种包膜RNA病毒，尽管它们在兽医学中很重要，但特征却很差。马动脉炎病毒（EAV）是该家族的原型成员，可引起马的大量疾病，而猪繁殖与呼吸综合征病毒（PRRSV）是猪工业中最重要的病原体。PRRSV引起持续感染，除了其糖蛋白的高度可变性外，这也是从猪场消除该病毒的主要障碍。持久的一个可能的分子原因是中和抗体出现较晚，但其蛋白质靶点和表位尚未确定。动脉病毒的包膜包含两个糖蛋白尖峰，Gp5/M和Gp2/3/4，它们存在于内质网或早期高尔基体（病毒颗粒的组装位点）中。反向遗传实验表明，芽殖需要Gp5/M，细胞进入需要Gp2/3/4。Gp2/3/4应该能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且基本上不为人所知的过程组装起来的。Gp2和Gp4在细胞内形成二硫化物连接的二聚体，可能是在内质网中，而Gp3和Gp2/4之间的二硫化物连接仅在出芽病毒颗粒中形成。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裂解。这些研究也导致了Gp3膜拓扑结构的新模型：未裂解的信号肽不作为膜锚，而是完全转移到内质网的管腔中。锚定是由疏水c端引起的，它不是跨膜区域，而是将Gp3附着在膜的周围。在我们的研究项目中，我们希望揭示Gp2/3/4的组装途径，并创建一个工具来分析其功能。我们将首先分析来自不同PRRSV菌株的Gp3是否遵循相同的独特加工方案，并表现出与来自EAV的Gp3相同的膜拓扑结构。有趣的是，PRRSV菌株Gp3的c端生物物理特性不同，这表明在一些菌株中Gp3可能不是膜结合的，而是从细胞分泌的。反向遗传学将用于分析Gp3的膜锚定对病毒传染性的意义。然后，我们将利用其组分在转染的细胞中组装天然的Gp2/3/4复合体。去除保留信号，这些信号尚未被识别，但可能位于蛋白质的跨膜区域，将允许复合物靶向质膜。暴露在表面的Gp2/3/4复合体适用于多种检测方法，如与细胞受体的附着、与抗体的结合和膜融合试验，因此有助于阐明Gp2/3/4尖峰在病毒进入过程中的作用，并作为中和抗体的假定靶点。

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.