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，必须首先与其细胞受体结合。Oldstone实验室分离并表征了包括拉沙热病毒（LFV）和LCMV C113在内的几种沙粒病毒的细胞受体α-肌营养不良聚糖（α-DG）。我们提供了α-DG是感染所需的受体的证据，因为：1）LFV和LFV糖蛋白（GP）以高亲和力与固定在膜上的纯化的α-DG结合; 2）携带DG基因无效突变的正常容许细胞对LFV感染具有抗性; 3）使用腺病毒载体在无效突变细胞中重建α-DG表达恢复对LFV的易感性。我们重新设计了一种高输出检测方法，用于分析BSL 2条件下LFVGP介导的靶细胞感染。在Boger实验室中，溶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鉴定促进蛋白质-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）的治疗化合物。 我们提出了三个具体的目标：第一，筛选组合化学库，发现小分子抑制LFVGP介导的感染细胞使用逆转录病毒载体，包含LFVGP在其包膜。第二，通过工程化抗病毒受体体开发中和游离LFV的药物，其中IgG分子的Fab部分被病毒结合的α-DG片段取代。第三，通过在小鼠模型中阻断LCMV C113感染（C113和LFV以高亲和力与a-DG结合并可预测地利用相同的结合位点）来验证这种体内抑制，最终验证将在CDC使用实验确定的化学抑制剂和毒性LFV进行。 研究科审查的初步赠款指出，该提案非常重要，令人兴奋和创新。然而，也注意到缺乏初步数据的弱点。为了纠正这种缺陷，我们1）改变了我们的高通量方法以使用荧光素酶报告系统，2）提供数据以显示我们可以抑制病毒结合而不抑制层粘连蛋白结合，3）提供构建抗病毒受体体的证据。

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.