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Analysis of localization, topology, membrane binding, secretion and function of the pestivirus structural glacoprotein Erns

瘟病毒结构糖蛋白 Erns 的定位、拓扑、膜结合、分泌和功能分析

基本信息

批准号：
16994418
负责人：
Professor Dr. Gregor Meyers
金额：
--
依托单位：
Friedrich-Loeffler-Institut - Bundesforschungsinstitut für Tiergesundheit (FLI)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2005
资助国家：
德国
起止时间：
2004-12-31 至 2015-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/16994418?language=en
关键词：
Analysis localization topology membrane binding

项目摘要

Pestiviruses belong to the family Flaviviridae and produce enveloped virions with a single stranded RNA genome of positive polarity. They have established fascinating mechanisms for establishment of long lasting persistence and are of major importance for veterinary medicine (Lindenbach et al, 2007).Pestivirus particles contain glycoprotein Erns that represents a crucial factor for virulence and persistence of pestivirus. It possesses RNase activity (Hulst & Moormann, 2001; Hulst et al, 1998; Schneider et al, 1993; Windisch et al, 1996), which we identified as virulence factor and cofactor for establishment of persistence (Meyer et al, 2002; Meyers et al, 1999; Tews et al, 2009; Meyers et al, 2007). Erns contains no typical membrane anchor and is partially secreted from infected cells (Magkouras et al, 2008; Mätzener et al, 2009; Rümenapf et al, 1993) whereas the overwhelming amount of the synthesized protein remains membrane bound within the ER or an ER-related compartment (Burrack et al, 2012; Tews & Meyers, 2007; Tews et al, 2009). The carboxyterminal region of Erns represents an unusual membrane anchor dar (Tews & Meyers, 2007; Tews et al, 2009), ensures the intracellular localization of the protein, the biologically important homodimerization and the processing of the Erns/E1 precursor. We could recently show that the Erns membrane anchor folds into an amphipathic helix in contact with a membrane. Unexpectedly the central part of this helix is located within the lipid bilayer. It contains a series of charged residues that could form a so-called charge zipper motif (Walther et al, 2013). The mechanism of Erns membrane anchoring and the function of the charge zipper motif in membrane anchoring will be analyzed with molecular techniques. Further structure analysis of the membrane bound anchor will be conducted in cooperation. The virulence factor function of Erns is dependent on the RNase activity and homodimer formation. We have isolated from animals a pseudorevertant that regained the ability to form dimers by a mutation in the middle of the membrane anchor. This finding raises questions concerning the primary sequence requirements and timely coordination of dimer formation, Erns/E1 processing and initiation of membrane binding, which will be investigated during the coming grant period. Erns secretion is hypothesized to be curcial for ist function as a virulence factor. We aim to analyze how the equilibrium between specific intracellular localization and secretion of the protein is achieved. Moreover, the role of the charge zipper motif for the insertion of the Erns carboxyterminus into the membrane and the influence of other viral proteins on Erns retention and recruitment into the virus particle will be investigated.

鼠疫病毒属于黄病毒科，产生具有正极性单链RNA基因组的包膜病毒粒子。它们已经建立了令人着迷的机制，以建立持久的持久性，对兽医学具有重要意义（Lindenbach et al, 2007）。鼠疫病毒颗粒中含有糖蛋白Erns，这是决定鼠疫病毒毒力和持久性的关键因素。它具有RNase活性（Hulst & Moormann, 2001； Hulst等人，1998；Schneider等人，1993；Windisch等人，1996），我们认为这是建立持久性的毒力因子和辅助因子（Meyer等人，2002；Meyers等人，1999；Tews等人，2009；Meyers等人，2007）。Erns不含典型的膜锚，部分由受感染细胞分泌（Magkouras等人，2008；Mätzener等人，2009;r<s:1> menapf等人，1993），而大量合成的蛋白仍在内质网或内质网相关的室内保持膜结合（Burrack等人，2012;Tews & Meyers, 2007； Tews等人，2009）。Erns的羧基末端区域代表了一个不寻常的膜锚区（Tews & Meyers, 2007; Tews et al, 2009），确保了蛋白质的细胞内定位、生物学上重要的同二聚化和Erns/E1前体的加工。我们最近可以证明，厄恩膜锚折叠成一个与膜接触的两性螺旋。出乎意料的是，这个螺旋的中心部分位于脂质双分子层内。它包含一系列带电残基，可以形成所谓的电荷拉链基序（Walther et al ., 2013）。本文将运用分子技术分析Erns膜锚定机理及电荷拉链基序在膜锚定中的作用。进一步的膜绑定锚的结构分析将在合作中进行。Erns的毒力因子功能依赖于RNase活性和同型二聚体的形成。我们已经从动物身上分离出一种假逆转物，它通过在膜锚的中间发生突变而重新获得了形成二聚体的能力。这一发现提出了有关二聚体形成、Erns/E1加工和膜结合起始的初级序列要求和及时协调的问题，这些问题将在即将到来的拨款期间进行研究。Erns分泌被认为是作为一种毒力因子而发挥作用的关键。我们的目的是分析如何平衡特定的细胞内定位和蛋白质的分泌是实现的。此外，电荷拉链基序在Erns羧基端插入膜中的作用以及其他病毒蛋白对Erns保留和招募到病毒颗粒中的影响将被研究。