ISG CONTROL OF FLAVIVIRUS INFECTION

描述（由申请人提供）：黄病毒属的成员是最重要的节肢动物传播的病毒，可引起人类疾病。该属包括在世界新地区重新出现并成为地方病的病毒（西尼罗病毒（WNV）、日本脑炎病毒（JEV）和登革热病毒（DENV））。黄病毒每年造成约1亿例感染，其中数十亿人处于危险之中，目前尚无专门的治疗方法。尽管干扰素（IFN）反应控制着西尼罗河病毒和其他黄病毒的细胞和组织趋向性，但限制感染的特定效应分子仍然缺乏特征。在Diamond和Chanda实验室之间的这个跨学科合作项目中，研究将使用遗传筛选来识别在体内和体外调节特定细胞类型黄病毒感染的新型干扰素刺激基因（ISG）。将使用定制生成的gfp标记shRNA文库进行功能缺失高通量遗传筛选，目标是约700只小鼠和人类ISG，以鉴定限制西尼罗河病毒毒株和减毒株感染的新型ISG。使用靶向减少或异位表达候选ISG的稳定细胞系，我们将从机制上定义新的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，我们已经确定了几种候选抑制ISG (ifif27、IFIT2和IFITM3)，它们可以减轻西尼罗河病毒感染，并且已经获得或产生了敲除小鼠。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isg具有抗病毒特性，以不同的方式控制西尼罗河病毒的感染和传播，并且这些表现出组织和细胞类型的特异性。总的来说，这些实验将更清楚地定义IFN控制与黄病毒发病机制之间的接口，并可能指导调节对该病毒家族感染的免疫策略。

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.