ISG CONTROL OF FLAVIVIRUS INFECTION

描述（由申请方提供）：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）和登革热病毒（DENV）），这些病毒在世界新地区重新出现并成为地方病。黄病毒每年造成约1亿例感染，数十亿人处于危险之中，并且没有特定的治疗方法。虽然干扰素（IFN）的反应控制的细胞和组织嗜性的西尼罗河病毒和其他黄病毒，限制感染的特定效应分子仍然很差的特点。Diamond和Chanda实验室之间的这项合作和跨学科项目中的研究将使用遗传筛选来鉴定新型干扰素刺激基因（ISG），这些基因在体外和体内调节特定细胞类型中的黄病毒感染。将使用定制生成的GFP标记的shRNA文库（靶向约700个小鼠和人ISG）进行功能丧失高通量遗传筛选，以鉴定限制WNV强毒株和减毒株感染的新型ISG。使用具有候选ISG的靶向减少或异位表达的稳定细胞系，我们将从机制上定义新型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，我们已经鉴定了几种减弱WNV感染的候选抑制性ISG（IFi27、IFIT2和IFITM3），并且已经获得或产生敲除小鼠。使用这些小鼠，我们将评估这些特定的ISG在限制WNV复制的细胞特异性方式和体内的功能。我们假设特异性ISGs具有差异控制西尼罗河病毒感染和传播的抗病毒特性，并且这些表现出组织和细胞类型特异性。总体而言，这些实验将更清楚地定义IFN控制和黄病毒发病机制之间的接口，并可能，指导策略，调节免疫力感染这一家族的病毒。

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.