Identification of Borna Disease Virus Receptor Proteins

描述（由申请方提供）：博尔纳病病毒（BDV）是一种具有非节段负链（NNS）RNA基因组的包膜病毒。由于其独特的特性，BDV是一个新病毒家族的原型成员。BDV具有高度嗜神经性，是研究CMS病毒持久性的重要模型系统。BDV进入宿主细胞是由BDV表面糖蛋白（G）与迄今未鉴定的细胞受体蛋白的特异性相互作用介导的。关于BDV-受体相互作用的研究一直受到感染性病毒粒子极度缺乏的阻碍。我们已经产生了重组VSV（rVSV.G*/p56），其中BDV G取代VSV G。值得注意的是，rVSV.G*/ p56重建了BDV的细胞嗜性和进入途径，并生长至高滴度。使用这种新的工具，我们现在可以实施新的策略来识别BDV细胞受体。我们的具体目标是： 1.鉴定BDV候选细胞受体蛋白。我们将使用遗传学和生物化学方法。遗传学方法将涉及受体无效细胞系与从对BDV高度易感的细胞和组织产生的cDNA文库的遗传互补。表达候选受体蛋白的互补细胞将通过它们对rVSV.G*/p56或BDV G-逆转录病毒假型的易感性来鉴定。生物化学方法将基于使用：（i）病毒覆盖蛋白印迹测定（VOPBA），和（ii）BDV G免疫粘附素，以选择与BDV G特异性相互作用的细胞表面蛋白。在这两种情况下，将使用MS程序确定蛋白质身份。 2. BDV候选细胞受体蛋白的功能验证。为此，我们将使用以下方法：1）用候选cDNA转染BDV受体缺失细胞应赋予对rVSV.G*/p56的易感性; 2）RNAi介导的候选受体在BDV易感细胞中的敲低表达应赋予细胞对BDV和rVSV.G*/p56的抗性增加; 3)BDV候选受体的体内表达模式的确定。

DESCRIPTION (provided by applicant): Borna disease virus (BDV) is an enveloped virus with a non-segmented negative strand (NNS) RNA genome. Because of its unique features BDV is the prototypic member of a new virus family. BDV is highly neurotropic and is an important model system for the study of CMS viral persistence. BDV entry into host cells is mediated by specific interactions of the BDV surface glycoprotein (G) with so far unidentified cellular receptor proteins. Studies about BDV-receptor interactions have been impeded by an extreme paucity of infectious virions. We have generated a recombinant VSV (rVSV.G*/p56) where the BDV G was substituted for the VSV G. Notably, rVSV.G*/ p56 recreates the cell tropism and entry pathway of BDV, and grows to high titers. Using this novel tool we can now implement new strategies by which to identify BDV cellular receptors. Our specific aims are: 1. Identify BDV candidate cellular receptor proteins. We will use genetic and biochemical approaches. The genetic approach will involve genetic complementation of receptor-null cell lines with cDNA libraries generated from cells and tissues that are highly susceptible to BDV. Complemented cells expressing candidate receptor proteins will be identified by their susceptibility to rVSV.G*/p56, or BDV G-retroviral pseudotypes. The biochemical approach will be based on the use of: (i) a virus overlay protein blot assay (VOPBA), and (ii) BDV G immunoadhesins to select cell surface proteins that specifically interact with BDV G. In both cases protein identities will be determined using MS procedures. 2. Functional validation of BDV candidate cellular receptor proteins. For this we will use the following approaches: 1) transfection of BDV receptor-null cells with the candidate cDNA should confer susceptibility to rVSV.G*/p56; 2) RNAi-mediated knock-down expression of candidate receptors in BDV susceptible cells should confer cells with increased resistance to both BDV and rVSV.G*/p56; 3) Determination of expression pattern of BDV candidate receptors in vivo.