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导致疾病，必须首先与其细胞受体结合。Oldstone实验室分离并鉴定了包括拉沙热病毒(Lassa First Virus，LFV)和LCMV CI 13在内的几种阿拉伯病毒的细胞受体α-Dstroglan(α-DG)。我们提供的证据表明，α-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的α-DG高亲和力结合；2)DG基因零突变的正常允许细胞对LFV感染具有抵抗力；3)用腺病毒载体在零突变细胞中重建α-DG的表达，恢复了对LFV的敏感性。我们重新设计了一种高产量的方法来分析BSL2条件下LFVGP介导的靶细胞感染。在博格实验室，溶液相合成技术被用来创建独特的小分子组合库，可以筛选出促进蛋白质-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。 我们提出了三个具体的目标：第一，筛选组合化学文库，以发现抑制LFVGP介导的细胞感染的小分子，使用在其包膜中含有LFVGP的逆转录病毒载体。第二，开发一种药物，通过设计抗病毒受体小体来中和游离的LFV，其中免疫球蛋白分子的Fab部分被病毒结合的α-DG片段取代。第三，为了在体内验证这种抑制作用，在小鼠模型中阻断LCMVCI 13感染(CI 13和LFV与α-DG高亲和力结合，并可预测地使用相同的结合部位)，最终验证将在疾控中心使用实验定义的化学抑制剂(S)和强毒LFV进行。研究科审查的最初赠款指出，该提案意义重大、令人振奋和具有创新性。然而，人们注意到了缺乏初步数据的弱点。为了纠正这一缺陷，我们1)改变了我们的高通量方法，使用了荧光素酶报告系统，2)提供了数据，表明我们可以在不抑制层粘连蛋白结合的情况下抑制病毒结合，3)提供了构建抗病毒受体小体的证据。

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.