Cell-free VEE assembly system and alphavirus drug screen

描述(申请人提供)：委内瑞拉马脑炎病毒(VEEV)引起高死亡率的神经系统疾病流行，并可通过气雾剂传播。因此，它被归类为NIAID B类优先病原体。目前，还没有获得许可的抗病毒药物用于治疗VEEV或其他甲型病毒。鉴于甲型病毒引起的疾病的高死亡率，在大规模生物恐怖袭击的情况下，开发用于暴露前和暴露后预防的抗病毒药物是重要的。在这些药物开发出来之前，治疗、疗效和递送问题是无法解决的。在这里，我们建议验证一种无细胞的VEEV衣壳组装系统，该系统可以在未来开发为鉴定组装抑制剂的高通量筛选。最近对其他病毒的研究证实了衣壳组装的抑制剂可能是有效的抗病毒药物。在这项应用中，我们提供了初步数据，表明VEEV衣壳在无细胞系统中高效组装。我们建议通过以下方法验证我们的发现：1)使用透射电子显微镜和其他分析方法来证实在无细胞系统中制造的VEEV衣壳与真实的衣壳非常相似；2)使用突变分析来定义组装所需的VEEV衣壳蛋白(CP)的结构域；以及3)使用RNA封装研究来定义VEEV包装信号。 我们的团队在开发无细胞衣壳组装系统方面拥有丰富的经验。对于拟议的研究，我们将与开发VEEV复制子系统的USAMRIID的研究人员合作。此外，我们与一家拥有化合物文库的小公司合作，一旦我们的检测方法得到这里提出的研究的验证，它将用于高通量筛选。无细胞VEE组装系统为了解VEE衣壳的形成机制和筛选具有潜在生物恐怖主义作用的甲型病毒活性物质提供了一种安全和低成本的方法。

DESCRIPTION (provided by applicant): Venezuelan equine encephalitis virus (VEEV) causes epidemics of neurological disease with high mortality, and can be spread by aerosol transmission. Thus, it has been categorized as an NIAID Class B priority pathogen. Currently, no licensed anti-viral agents exist for treatment of VEEV or other alphaviruses. Development of anti-viral agents for pre-exposure and post-exposure prophylaxis in the case of a large scale bioterrorist attack is important, given the high fatality rate of the disease caused by this alphavirus. The window of treatment, efficacy, and delivery issues cannot be addressed until such drugs are developed. Here we propose to validate a cell-free system for VEEV capsid assembly that can be developed in the future as a high throughput screen for identification of assembly inhibitors. Recent studies with other viruses have validated Inhibitors of capsid assembly potentially efficacious anti-viral drugs. In this application, we provide preliminary data indicating highly efficient assembly of VEEV capsids in the cell-free system. We propose to validate our findings by: 1) using transmission electron microscopy and other assays to confirm that the VEEV capsids made in the cell-free system closely resemble authentic capsids; 2) using mutational analysis to define domains of VEEV capsid protein (CP) needed for assembly; and 3) using RNA encapsidation studies to define the VEEV packaging signal. Our group has extensive experience in developing cell-free capsid assembly systems. For the proposed studies we will collaborate with investigators at USAMRIID who developed the VEEV replicon system. In addition, we are partnered with a small company that has a compound library and will use our assay for high throughput screening, as soon as it is validated by the studies proposed here. The cell-free VEE assembly system offer a safe and low cost approach to understanding the mechanism of VEE capsid formation and to screen for agents active against alphaviruses that have potential as bioterrorism agents.