Cell-free VEE assembly system and alphavirus drug screen

描述（由申请方提供）：委内瑞拉马脑炎病毒（VEEV）引起神经系统疾病流行，死亡率高，可通过气溶胶传播。因此，它被归类为NIAID B类优先病原体。目前，不存在用于治疗VEEV或其他甲病毒的许可的抗病毒剂。鉴于这种甲病毒引起的疾病的高死亡率，在发生大规模生物恐怖袭击的情况下，开发用于接触前和接触后预防的抗病毒药物非常重要。在开发出此类药物之前，无法解决治疗窗口、疗效和交付问题。在这里，我们建议验证一个无细胞系统的VEEV衣壳组装，可以在未来开发为一个高通量筛选识别组装抑制剂。最近对其他病毒的研究已经验证了衣壳组装抑制剂潜在有效的抗病毒药物。在本申请中，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测定法来确认在无细胞系统中制备的VEEV衣壳与真实衣壳非常相似; 2）使用突变分析来定义组装所需的VEEV衣壳蛋白（CP）结构域; 3）使用RNA水解研究来定义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USAMRIID的研究人员合作，他们开发了VEEV复制子系统。此外，我们与一家拥有化合物库的小公司合作，一旦通过本文提出的研究验证，将使用我们的检测方法进行高通量筛选。无细胞VEE组装系统提供了一种安全且低成本的方法来理解VEE衣壳形成的机制，并筛选对具有生物恐怖主义剂潜力的甲病毒具有活性的试剂。

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.