The role of the GP5-M Spike of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) for virus budding and viral persistence

猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）是动脉炎病毒科（Arteriviridae）的一种负链RNA病毒，是养猪业中最重要的病原体。与M蛋白形成二硫键连接的糖蛋白5是主要的包膜蛋白，对于病毒出芽和重要的抗体靶标是必需的。PRRSV引起持续感染，除了其糖蛋白的高度变异性之外，这是从猪群中消除病毒的主要障碍。关于持续性的机制基础的一种假说，即诱饵假说，假定在病毒复制的初始阶段逃避适应性免疫应答允许建立持续性感染。根据这个模型，抗GP 5的抗体在感染后不久产生，但不能中和病毒。中和抗体在感染后几周内出现，最终清除病毒。GP 5上两种类型抗体的表位彼此非常接近，由具有相等可变数量的碳水化合物的高变区隔开。提出非中和抗体的结合位点是诱饵表位，针对其制备抗体以防止中和抗体的产生或结合。然而，诱骗表位位于信号肽中，信号肽是GP 5靶向所需的分子结构域。在上一个基金期间，我们发现信号肽从各种PRRSV株的GP 5上被切割，不依赖于其附近的碳水化合物。通过质谱鉴定了2型PRRSV株GP 5的两个信号肽切割位点。可变切割导致在病毒颗粒中产生两个GP 5蛋白群体，一个不含诱饵表位，另一个含诱饵表位。他的建议的第一个目标是产生含有仅在位点1或位点2切割的GP 5的重组PRRSV。然后，所得的病毒体含有GP 5分子的同质群体，完全缺乏或保留诱饵表位。在随后的实验中，重组病毒将用于感染仔猪，以检验诱饵假说。第二个目的是功能性地探索包括GP 5-M作为其中心元件的PRRSV的出芽。将分析GP 5和M的共表达是否诱导VLP的形成，VLP是包含包埋在膜中的病毒蛋白并具有与真实病毒体相同的密度和大小的颗粒。然后，我们将研究GP 5-M的胞质尾是否与N蛋白相互作用，以招募病毒基因组到出芽位点。我们将确定GP 5和M中的近膜半胱氨酸是否被棕榈酰化，以及这种修饰是否诱导GP 5-M寡聚体的形成作为病毒出芽的驱动力。最后，通过表达VPS的显性阴性突变体，我们将确定细胞ESCRT模块是否是释放病毒颗粒所必需的。

The porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped negative stranded RNA-virus in the Arteriviridae family, is the most important pathogen in the porcine industry. Glycoprotein 5 that forms a disulphide-link with the M protein is the main envelope protein, essential for virus budding and an important antibody target. PRRSV causes persistent infection, which is, besides the high variability of its glycoproteins, the main obstacle to elimination of the virus from pig populations. One hypotheses on the mechanistic basis for persistence, the decoy hypothesis, posits that evasion of adaptive immune responses during the initial phase of virus replication allows establishment of persistent infection. According to this model, antibodies against GP5 are made shortly after infection but do not neutralize the virus. Neutralizing antibodies appear only weeks into the infection to finally clear the virus. The epitopes of both types of antibodies on GP5 are located in close proximity to each other, separated by a hypervariable region with an equally variable number of carbohydrates. The binding site for the non-neutralizing antibodies was proposed to be a decoy epitope, against which antibodies are made that prevent the generation or binding of neutralizing antibodies. However, the decoy epitope is located in the signal peptide, a molecular domain required for targeting of GP5.In the last funding period we showed that the signal peptide is cleaved from GP5 of various PRRSV strains, independent of carbohydrates in its vicinity. Two signal peptide cleavage sites were identified for GP5 of genotype 2 PRRSV strains by mass spectrometry. Variable cleavage results in the production in two populations of GP5 proteins in virus particles, one without and the other with the decoy epitope. The first objective of his proposal is to generate recombinant PRRSV that contains GP5 cleaved only at site 1 or at site 2. The resulting virions then contain a homogenous population of GP5 molecules, either completely lacking or retaining the decoy epitope. In subsequent experiments recombinant viruses will be used to infect piglets to test the decoy hypothesis. The second objective is to functionally explore budding of PRRSV that includes GP5-M as its central element. It will be analyzed whether co-expression of GP5 and M induces the formation of VLPs, particles containing the viral proteins embedded in a membrane and having the same density and size as authentic virions. We will then investigate whether cytoplasmic tails of GP5-M interact with the N protein to recruit the viral genome to the budding site. We will determine whether membrane-proximal cysteines in GP5 and M are palmitoylated and whether this modification induces the formation of GP5-M oligomers as the driving force for virus budding. Finally, by expression of a dominant-negative mutant of VPS, we will determine if the cellular ESCRT module is required for release of virus particles.