Development of HTS Assays for Inhibitors of Viral-Cell Interactions

描述(申请人提供)：汉坦病毒会导致严重的人类疾病：肾综合征出血热(HFRS)和汉坦病毒心肺综合征(HCPS)。缺乏预防或治疗HCPS的疫苗或特定药物，以及要求在生物安全3级实验室(BSL-3)进行实验，限制了探索感染机制和疾病发病机制的能力。病死率一般在30%至50%之间。目前在美国还没有疫苗或特定的治疗方法可供使用。高病死率，缺乏疫苗和治疗方法，以及汉坦病毒能够通过气溶胶途径传播，导致它们被归类为A类病原体和潜在的生物战病原体。因此，我们启动了一个项目，研究校准剂量的紫外线辐射杀死的新诺布雷病毒(SNV)，作为一个模型系统，以剖析其进入BSL-2设施的细胞机制。我们证明了紫外线灭活的SNV与糖基磷脂酰肌醇(GPI)锚定的蛋白衰变加速因子(DAF/CD55)和低亲和力状态的avb3整合素以类似于活病毒粒子的方式附着。我们建议通过阻断汉坦病毒与其表面共受体DAF的结合和通过？v？进入细胞来筛选汉坦病毒感染的小分子抑制物。整合素。为了实现这些目标，我们将追求以下目标：目标1.开发与新诺布病毒DAF和？V？结合的小分子抑制剂的HTS初步筛选。目的2.建立SNV细胞相互作用小分子抑制物二次筛选方法。 公共卫生相关性：拟议的工作使用病毒学和定量生物物理工具的独特跨学科组合来确定致病汉坦病毒的小分子抑制物，这些小分子抑制物会导致汉坦病毒心肺综合征(HCPS)和肾综合征出血热(HFRS)。目前，美国还没有可用于治疗猪瘟或肾综合征出血热的疫苗或特定疗法。高病死率、缺乏疫苗和治疗方法导致它们被归类为A类病原体和潜在的生物战因素。我们的初步研究表明，紫外线灭活的病毒与活病毒使用的受体结合。因此，我们将建立与流式细胞术兼容的高通量筛选方法，用于汉坦病毒与其表面共受体衰变加速因子和进入受体？V？整合素。该项目的成功完成将导致治疗性干预的开发--针对汉坦病毒感染和依赖于这些受体进行感染的其他病原体的探针。

DESCRIPTION (provided by applicant): Hantaviruses cause severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HCPS, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. Case fatality ratios generally range from 30 to 50%. No vaccines or specific therapy are currently available for use in the US. The high case-fatality rate, the absence of vaccines and therapeutics, and the ability of Hantaviruses to be transmitted via the aerosol route have led to their categorization as Category A pathogens, and potential agents of biological warfare. We therefore initiated a program to study Sin Nombre virus (SNV) killed with a calibrated dose of UV radiation as a model system to dissect its mechanism of cellular entry in BSL-2 facilities. We demonstrated that UV-killed SNV attaches to the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF/CD55) and low affinity state avb3 integrins in a manner that parallels live virions. We propose to screen for small molecule inhibitors of hantavirus infection by blocking the binding of hantaviruses to their surface co- receptor DAF and cellular entry through ?v?? integrins. To achieve these goals, we will pursue the following aims: Aim 1. To develop HTS primary screens for small molecule inhibitors of binding of Sin Nombre Viruses to DAF and ?v??. Aim 2. To develop secondary HT screening assays for small molecule inhibitors of SNV cell interactions. PUBLIC HEALTH RELEVANCE: The proposed work uses a unique interdisciplinary combination of virology and quantitative biophysical tools to identify small molecule inhibitors of pathogenic hantaviruses, which cause hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). There are currently no vaccines or specific therapy that are available for use in the US to treat HCPS or HFRS. The high case-fatality rate, the absence of vaccines and therapeutics has led to their categorization as Category A pathogens, and potential agents of biological warfare. Our preliminary studies have shown that UV-killed viruses bind to the same receptors used by live viruses. Thus we will develop flow cytometry-compatible high throughput screening assays for small molecule inhibitors of the binding of hantaviruses to its surface co-receptor Decay Accelerating factor and entry receptor ?v?? integrin. Successful completion of this project will lead to the development of therapeutic intervention-probes for hantavirus infection and other pathogens that rely on these receptors for infection.