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.
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| Clemente, Roberto; de Parseval, Aymeric; Perez, Mar et al. (2009) Borna disease virus requires cholesterol in both cellular membrane and viral envelope for efficient cell entry. J Virol 83:2655-62 |
| Perez, Mar; Clemente, Roberto; Robison, Clinton S et al. (2007) Generation and characterization of a recombinant vesicular stomatitis virus expressing the glycoprotein of Borna disease virus. J Virol 81:5527-36 |
| Clemente, Roberto; de la Torre, Juan C (2007) Cell-to-cell spread of Borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein. J Virol 81:5968-77 |
