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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 (PRRSV) 的 GP5-M 刺突对病毒出芽和病毒持久性的作用

基本信息

批准号：
193516483
负责人：
Professor Dr. Nikolaus Osterrieder
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：
2010
资助国家：
德国
起止时间：
2009-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/193516483?language=en
关键词：
role GP5 Spike Porcine Reproductive

项目摘要

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.

猪繁殖与呼吸综合征病毒（PRRSV）是猪动脉病毒科的一种包膜负链rna病毒，是养猪业中最重要的病原体。糖蛋白5与M蛋白形成二硫键，是病毒出芽所必需的主要包膜蛋白，也是重要的抗体靶点。PRRSV引起持续感染，除了其糖蛋白的高度可变性外，这也是从猪群中消除该病毒的主要障碍。一种基于持续性机制的假说，即诱饵假说，认为在病毒复制的初始阶段，逃避适应性免疫反应可以建立持续性感染。根据这个模型，针对GP5的抗体在感染后不久就会产生，但不会中和病毒。中和抗体在感染几周后出现，最终清除病毒。GP5上的两种抗体的表位彼此靠近，由一个具有相同可变数量的碳水化合物的高可变区域隔开。非中和抗体的结合位点被认为是一个诱饵表位，针对它产生的抗体可以阻止中和抗体的产生或结合。然而，诱饵表位位于信号肽中，这是靶向GP5所需的分子结构域。在上一个资助期，我们证明了信号肽是从各种PRRSV菌株的GP5中切割出来的，独立于其附近的碳水化合物。用质谱法鉴定了基因2型PRRSV株GP5的两个信号肽切割位点。可变切割导致病毒颗粒中产生两种GP5蛋白，一种不含假抗原表位，另一种含假抗原表位。他建议的第一个目标是产生重组PRRSV，其中包含仅在位点1或位点2切割的GP5。然后产生的病毒粒子含有均匀的GP5分子群，要么完全缺乏，要么保留诱饵表位。在随后的实验中，将使用重组病毒感染仔猪来验证诱饵假说。第二个目标是功能性地探索包括GP5-M作为其中心元素的PRRSV的出芽。将分析GP5和M的共表达是否诱导了VLPs的形成，VLPs是一种包含病毒蛋白的颗粒，嵌入在膜上，具有与真实病毒粒子相同的密度和大小。然后，我们将研究GP5-M的细胞质尾部是否与N蛋白相互作用，将病毒基因组招募到出芽位点。我们将确定GP5和M的近端半胱氨酸是否棕榈酰化，以及这种修饰是否诱导GP5-M寡聚物的形成，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模块是否需要释放病毒颗粒。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus.

DOI：
10.1016/j.virusres.2017.08.004
发表时间：
2017-08
期刊：
Virus research
影响因子：
5
作者：
B. Thaa;Susanne Kaufer;S. A. Neumann;Bernadett Peibst;H. Nauwynck;E. Krause;M. Veit
通讯作者：
B. Thaa;Susanne Kaufer;S. A. Neumann;Bernadett Peibst;H. Nauwynck;E. Krause;M. Veit

Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae.