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

动脉炎病毒科是一类有包膜 RNA 病毒，尽管它们在兽医学中很重要，但对其特征却知之甚少。马动脉炎病毒（EAV）是该家族的原型成员，可引起马的严重疾病，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。 PRRSV造成持续感染，除了其糖蛋白的高度变异性之外，这也是猪场消除该病毒的主要障碍。持久性的一个可能的分子原因是中和抗体的较晚出现，但其蛋白质靶点和表位尚未确定。动脉病毒的包膜含有两个糖蛋白刺突，Gp5/M 和 Gp2/3/4，它们位于内质网或早期高尔基体（病毒颗粒的组装位点）中保留的细胞中。反向遗传实验表明出芽需要 Gp5/M，细胞进入需要 Gp2/3/4。 Gp2/3/4 应该将动脉病毒附着在细胞受体上并催化膜融合。二硫键连接的 Gp2/3/4 刺突是通过复杂且很大程度上不被理解的过程组装的。 Gp2 和 Gp4 在细胞内（可能是在内质网中）形成二硫键连接的二聚体，而 Gp3 和 Gp2/4 之间的二硫键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。这些研究还产生了 Gp3 膜拓扑的新模型：未切割的信号肽不充当膜锚，而是完全易位到 ER 腔中。锚定是由疏水性 C 末端引起的，它不是跨膜区域，但将 Gp3 附着在膜的外围。通过我们的研究项目，我们希望解开 Gp2/3/4 的组装途径，并创建一个工具来分析其功能。我们首先应分析来自各种 PRRSV 菌株的 Gp3 是否遵循与来自 EAV 的 Gp3 相同的独特处理方案并表现出相同的膜拓扑结构。有趣的是，PRRSV 菌株的 Gp3 C 末端的生物物理特性有所不同，这表明在某些菌株中，Gp3 可能不是膜结合的，而是从细胞中分泌的。将利用反向遗传学分析Gp3膜锚定对病毒感染性的意义。然后我们将在转染细胞中用其成分组装天然 Gp2/3/4 复合物。保留信号尚未被识别，但可能位于蛋白质的跨膜区域，去除保留信号将允许复合物靶向质膜。表面暴露的 Gp2/3/4 复合物适用于多种检查方法，例如与细胞受体的附着、与抗体的结合和膜融合测定，因此有助于阐明 Gp2/3/4 刺突在病毒进入过程中的作用以及作为中和抗体的推定靶标。

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.