Characterization of the non-structural protein p4 of the recently detected new virus European mountain ash ringspot-associated virus in European mountain ash (Sorbus aucuparia L.)

欧洲山地水曲柳环斑相关病毒(EMARaV)侵染欧洲水曲柳(Sorbus ucuparia L.)并广泛分布在北欧和中欧。它会导致植物从退化到死亡。到目前为止，EMARaV的传播方式、可能的寄主范围和经济意义尚不清楚。我们将EMARaV描述为一种新型的负定向单链RNA病毒，可通过嫁接传播(Führling和Büttner，1995)。已识别的四种病毒RNA中的每一种都含有一个开放阅读框架。序列比较表明，RNA1、RNA2和RNA3编码的蛋白质可能分别作为RNA依赖的RNA聚合酶(P1)、糖蛋白前体(P2)和核衣壳蛋白(P3)发挥功能。到目前为止，RNA4编码的p4蛋白的功能尚不清楚。该RNA的开放阅读框架没有显示出与已知蛋白质的序列同源性。我们推测该蛋白可能作为基因沉默抑制子和/或运动蛋白。在植物中，EMARaV的细胞间传播和系统分布需要病毒运动蛋白。在这个提案中，我们旨在研究p4蛋白可能的运动功能。我们将使用p4特异性抗体在p4转基因的原生质体和感染EMARaV的欧洲山灰树叶的横切面中鉴定p4蛋白的定位。我们将研究p4是否利用特定的胞间连丝标记与细胞壁共定位。EMARaV可能作为核衣壳包裹的基因组RNA(核糖核蛋白复合体)沿着病毒运动蛋白聚集形成的p4管状结构移动。我们将检查在p4转基因的原生质体中是否发生小管形成。小管的形成和核糖核蛋白复合体沿这些结构的运输将在蛋白质-蛋白质相互作用实验中得到验证。我们将使用双分子荧光互补技术(BIFC)分析p4蛋白是否与自身和核衣壳蛋白相互作用。胆汁螨已被确定为该病毒的潜在载体，EMARaV可能在该载体中复制。在蚊子中检测到非结构蛋白p4将进一步证实病毒以循环传播的方式传播。本研究的主要目的是调查和了解EMARaV p4蛋白的功能，从而加深我们对EMARaV传播和流行病学的迫切需要的知识。

The European mountain ash ringspot-associated virus (EMARaV) infects the European mountain ash (Sorbus aucuparia L.) and is widespread throughout Northern- and Central Europe. It leads from degeneration up to die back of the plant. EMARaV has been associated with the ringspot disease of Sorbus aucuparia L. So far, modes of virus transmission, possible host range and economic importance of EMARaV are unknown. We characterized EMARaV as a novel negative orientated single strand RNA virus that can be transmitted by grafting (Führling and Büttner, 1995). Each of the four identified viral RNAs contains one open reading frame. Sequence comparisons revealed a possible function of the RNA1, RNA2 and RNA3 encoded proteins as a RNA dependent RNA polymerase (p1), a glycoprotein precursor (p2) and a nucleocapsid protein (p3) respectively. So far, the function of the RNA4 encoded p4 protein is unknown. The open reading frame of this RNA shows no sequence homologies to known proteins. We hypothesize that this protein might act as a gene silencing suppressor and/or a movement protein. In plants, cell-to-cell spread as well as systemic distribution of EMARaV requires a viral movement protein. In this proposal we aim to investigate the possible movement function of the p4 protein.The localization of the p4 protein will be identified in p4-transfected protoplasts and in cross sections of EMARaV-infected European mountain ash leaves using a p4 specific antibody. We will investigate whether p4 colocalizes with cell walls taking advantage of specific plasmodesmal markers. EMARaV is presumably moving as nucleocapsid coated genomic RNA (ribonucleoprotein complex) along p4 tubular structures formed as a result of the aggregation of viral movement proteins. We will examine whether tubule formation takes place in p4-transfected protoplasts. The formation of tubuli and the transport of ribonucleoprotein complexes along those structures will be verified in protein-protein interaction experiments. We will analyze whether the p4 protein interacts with itself and nucleocapsid proteins using bimolecular fluorescence complementation (BIFC).The gall mite Phytoptus pyri has been identified as a potential vector of this virus and replication of EMARaV in this vector is likely. The detection of the non-structural protein p4 in the gall mite would further confirm virus transmission in a circulative propagative manner. The main objective of this study is to investigate and understand the function of the p4 protein of EMARaV and will thus deepen our much needed knowledge about spread and epidemiology of EMARaV.