Identification of Borna Disease Virus Receptor Proteins

描述(申请人提供)：博尔纳病病毒(BDV)是一种包膜病毒，具有非分段负链(NNS)RNA基因组。由于其独特的特征，BDV是一个新的病毒家族的原型成员。BDV具有高度的神经嗜性，是研究CMS病毒持久性的重要模型系统。BDV进入宿主细胞是通过BDV表面糖蛋白(G)与迄今尚未确定的细胞受体蛋白的特异性相互作用而介导的。有关BDV-受体相互作用的研究因极少的感染性病毒粒子而受到阻碍。我们已经构建了重组VSV(rVSV.G*/p56)，其中BDV G取代了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，并生长到高滴度。使用这个新的工具，我们现在可以实施新的策略，通过它来识别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。我们将使用遗传和生化方法。遗传方法将涉及受体缺失的细胞系与从对BDV高度敏感的细胞和组织产生的cDNA文库进行遗传互补。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-逆转录病毒假型的敏感性来鉴定。生化方法将基于：(I)病毒覆盖蛋白印迹分析(VOPBA)和(Ii)BDV G免疫粘附素来选择与BDV G特异相互作用的细胞表面蛋白。在这两种情况下，将使用MS程序确定蛋白质的身份。 2.BDV候选细胞受体蛋白的功能验证为此，我们将采用以下方法：1)将候选的BDV受体缺失的细胞与rVSV.G*/p56一起转染BDV受体阴性的细胞，使其对rVSV.G*/p56易感；2)RNAi介导的候选受体在BDV易感细胞中的下调表达，使细胞对BDV和rVSV.G*/p56的抵抗力增强； 3)BDV候选受体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.