Identification of Borna Disease Virus Receptor Proteins

描述（由申请人提供）：博纳病病毒（BDV）是一种包膜病毒，具有非节段负链（NNS） RNA基因组。由于其独特的特征，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进入宿主细胞是通过BDV表面糖蛋白(G)与迄今未识别的细胞受体蛋白的特异性相互作用介导的。传染性病毒粒子的极度缺乏阻碍了对bdv受体相互作用的研究。我们生成了重组VSV (rVSV.G*/p56)，其中BDV G取代了VSV G。G*/ p56重现了BDV的细胞趋向性和进入途径，并生长到高滴度。利用这个新工具，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：

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.