Therapeutics to Prevent/Treat Lassa Fever Virus

描述（由申请人提供）：病毒要感染细胞并随后引起疾病，必须首先与细胞受体结合。Oldstone实验室分离并鉴定了几种沙病毒（包括拉沙热病毒（LFV）和LCMV CI 13）的细胞受体α -三磷酸腺苷（α - dg）。我们提供的证据表明，a-DG是感染所需的受体，因为：1)LFV和LFV糖蛋白（GP）与固定在膜上的纯化α - dg具有高亲和力；2)携带DG基因零突变的正常允许细胞对LFV感染具有抗性；3)利用腺病毒载体重建零突变细胞中α - dg的表达，恢复对LFV的易感性。我们重新设计了一种高输出实验来分析BSL2条件下lfvgp介导的靶细胞感染。在Boger实验室，液相合成技术已被用于创建独特的小分子组合文库，这些小分子可以被筛选以识别促进蛋白质-蛋白质相互作用（激动剂）或抑制蛋白质-蛋白质相互作用（拮抗剂）的治疗性化合物。

DESCRIPTION (provided by applicant): For a virus to infect a cell and subsequently cause disease it must first bind to its cellular receptor. The Oldstone laboratory isolated and characterized the cellular receptor, alpha-dystroglycan (alpha-DG) for several arenaviruses including Lassa fever virus (LFV) and LCMV CI 13. We provided evidence that a-DG is the receptor required for infection as: 1) LFV and LFV glycoprotein (GP) bind at high affinity to purified alpha-DG immobilized on membranes; 2) normally permissive cells bearing a null mutation of the DG gene are resistant to LFV infection; 3) reconstitution of alpha-DG expression in null mutant cells using an adenovirus vector restored susceptibility to LFV. We have redesigned a high-output assay to analyze LFVGP-mediated infection of target cells under BSL2 conditions. In the Boger laboratory solution-phase synthetic techniques have been utilized to create unique combinatorial libraries of small molecules that can be screened to identify therapeutic compounds that promote protein-protein interactions (agonists) or inhibit protein-protein interactions (antagonists). We propose three specific aims: First, to screen combinatorial chemical libraries to discover small molecules that inhibit LFVGP-mediated infection of cells using retroviral vectors that contain LFVGP in their envelope. Second, to develop a drug that would neutralize free LFV by engineering anti-viral receptor-bodies, in which the Fab part of an IgG molecule is replaced by a virus-binding alpha-DG fragment. Third, to validate this inhibition in vivo by blocking LCMV CI 13 infection in a mouse model (CI 13 and LFV binds at high affinity to alpha-DG and predictably utilizes the same binding site) with final verification to be done at CDC using the experimentally defined chemical inhibitor(s) and virulent LFV. The initial grant reviewed by the Study Section noted the proposal to be highly significant, exciting and innovative. However, a weakness of a lack of preliminary data was noted. To correct this deficiency we have 1) altered our high-throughput approach to use a luciferase reporter system, 2) provide data to show we can inhibit virus binding without inhibition of laminin binding, 3) provide evidence of construction of antiviral receptor bodies.