Cell-free VEE assembly system and alphavirus drug screen

无细胞VEE组装系统和甲病毒药物筛选

• 批准号: 6976796
• 负责人: JAISRI R LINGAPPA
• 金额: $ 22.3万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6976796
• 关键词: Alphavirus; Venezuelan equine encephalitis virus; antiserum; antiviral agents; arthropod borne communicable disease; biotechnology; bioterrorism /chemical warfare; capsid; cell free system; cell line; clinical research; drug discovery /isolation; high throughput technology; laboratory rabbit; transmission electron microscopy; virus RNA; virus assembly; virus protein

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.

描述（由申请人提供）：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病，可通过气溶胶传播。因此，它已被归类为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。鉴于这种甲病毒引起的疾病的高致死率，在发生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非常重要。在开发出此类药物之前，无法解决治疗窗口期、疗效和递送问题。在这里，我们建议验证一种用于VEEV衣壳组装的无细胞系统，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抑制剂可能是有效的抗病毒药物。在这个应用中，我们提供了初步数据，表明在无细胞系统中VEEV衣壳的高效组装。我们打算通过以下方法来验证我们的发现：1)利用透射电子显微镜和其他实验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)利用突变分析确定VEEV衣壳蛋白（CP）组装所需的结构域；3)利用RNA封装研究来定义VEEV包装信号。