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

猪繁殖与呼吸综合征病毒 (PRRSV) Gp2/3/4 刺突的加工和功能

• 批准号: 256219832
• 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: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/256219832?language=en
• 关键词: Processing; Function; Gp2; Spike; Porcine

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.

动脉炎病毒科是一个包膜RNA病毒家族，尽管其在兽医学中很重要，但其特征很差。马动脉炎病毒（EAV）是该家族的原型成员，并在马中引起大量疾病，而猪繁殖与呼吸综合征病毒（PRRSV）是养猪业中最重要的病原体。猪繁殖与呼吸综合征病毒（PRRSV）的持续感染，除了其糖蛋白的高度变异性外，也是从猪场消除病毒的主要障碍。持久性的一个可能的分子原因是中和抗体的后期出现，但它们的蛋白质靶点和表位尚未确定。动脉炎病毒的包膜含有两个糖蛋白刺突，Gp 5/M和Gp 2/3/4，它们保留在ER或早期高尔基体（病毒颗粒的组装位点）中的细胞中。反向遗传实验表明，Gp 5/M是出芽所必需的，Gp 2/3/4是进入细胞所必需的。GP 2/3/4被认为是将动脉炎病毒附着到细胞受体上并催化膜融合。二硫键连接的Gp 2/3/4尖峰是通过一个复杂且很大程度上无法理解的过程组装的。而Gp 2和Gp 4在细胞内形成二硫键连接的二聚体，推测在ER中，Gp 3和Gp 2/4之间的二硫键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Gp 3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。这些研究也为Gp 3的膜拓扑结构提供了一个新的模型：未裂解的信号肽不作为膜锚，而是完全易位到ER的内腔中。疏水性C-末端不是跨膜区，但将Gp 3外周连接到膜上。通过我们的研究项目，我们希望解开Gp 2/3/4的组装途径，并创建一个分析其功能的工具。我们将首先分析来自各种PRRSV毒株的Gp 3是否遵循相同的独特加工方案并表现出与来自EAV的Gp 3相同的膜拓扑结构。有趣的是，PRRSV毒株在Gp 3的C-末端的生物物理性质上不同，这表明在一些毒株中Gp 3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Gp 3的膜锚定对病毒感染性的意义。然后，我们将在转染细胞中从其组分组装天然Gp 2/3/4复合物。去除保留信号（尚未鉴定，但可能位于蛋白质的跨膜区）将使复合物靶向质膜。表面暴露的Gp 2/3/4复合物适合于多种检查方法，例如附着于细胞受体、结合于抗体和膜融合测定，并且因此有助于阐明Gp 2/3/4刺突在病毒进入期间的作用以及作为中和抗体的推定靶标。