ISG CONTROL OF FLAVIVIRUS INFECTION

描述(申请人提供)：黄病毒属成员是引起人类疾病的最重要的节肢动物传播病毒。该属包括病毒(西尼罗河病毒(WNV)、日本脑炎病毒(JEV)和登革病毒(DENV))，它们正在重新出现并在世界新地区流行。黄病毒每年造成约1亿人感染，数十亿人面临风险，而且没有特定的治疗方法。尽管干扰素的反应控制着西尼罗河病毒和其他黄病毒的细胞和组织嗜性，但限制感染的特异性效应分子仍然缺乏特征。钻石实验室和昌达实验室之间的这一合作和跨学科项目中的研究将使用遗传筛选来识别新的干扰素刺激基因(ISG)，这些基因在体内和体外特定类型的细胞中调节黄病毒感染。针对大约700只小鼠和人类ISG的定制产生的GFP标记的shRNA文库将进行功能丧失的高通量遗传筛选，以识别限制西尼罗河病毒强毒株和弱毒株感染的新ISG。利用具有靶向减少或异位表达候选ISG的稳定细胞系，我们将从机械上定义新的ISG效应分子如何限制病毒生命周期中的特定步骤并影响感染结果。在初步研究中，我们已经确定了几个候选的抑制ISG(IFI27、IFIT2和IFITM3)，它们可以减弱WNV的感染，并且已经获得或产生了基因敲除小鼠。利用这些小鼠，我们将评估这些特定的ISG在细胞特异性方式和体内限制西尼罗河病毒复制方面的功能。我们假设特定的ISGs具有不同地控制西尼罗河病毒感染和传播的抗病毒特性，并且这些特性显示出组织和细胞类型的特异性。总体而言，这些实验将更清楚地定义干扰素控制和黄病毒致病机制之间的接口，并可能指导调节对这一病毒家族感染的免疫的策略。

DESCRIPTION (provided by applicant): Members of Flavivirus genus are the most important arthropod-borne viruses causing disease in humans. This genus includes viruses (West Nile virus (WNV), Japanese encephalitis virus (JEV) and Dengue virus (DENV)) that are re-emerging and becoming endemic in new areas of the world. Flaviviruses account for ~100 millions infections per year, with billions at risk and no specific therapy available. Although interferon (IFN) responses control the cell and tissue tropism of WNV and other flaviviruses, the specific effector molecules that restrict infection remain poorly characterized. The studies in this collaborative and inter-disciplinary project between the Diamond and Chanda laboratories will use genetic screens to identify novel interferon stimulated genes (ISG) that modulate flavivirus infection in specific cell types ex vivo and in vivo. Loss-of-function high-throughput genetic screens will be performed with a custom- generated GFP-marked shRNA library targeting ~700 mouse and human ISGs, to identify novel ISG that restrict infection of virulent and attenuated strains of WNV. Using stable cell lines that have targeted reductions or ectopic expression of candidate ISGs, we will define mechanistically how novel ISG effector molecules restrict specific steps in the viral lifecycle and influence infection outcome. In preliminary studies, we have identified several candidate inhibitory ISG (Ifi27, IFIT2, and IFITM3) that attenuate WNV infection, and already acquired or generated knockout mice. Using these mice, we will evaluate the function of these particular ISGs in restricting WNV replication in a cell-specific manner and in vivo. We hypothesize that specific ISGs have antiviral properties that differentially control WNV infection and spread, and that these demonstrate tissue and cell-type specificity. Overall, these experiments will more clearly define the interface between IFN control and flavivirus pathogenesis and possibly, guide strategies that modulate immunity to infection by this family of viruses.