Kianse Networks Controling Flavivirus Replication

黄病毒在其感染周期的每一步都需要并操纵宿主细胞途径。由于许多主机 细胞途径由蛋白激酶控制，这类宿主蛋白密切参与调节细胞内的蛋白质代谢。 黄病毒复制均为阴性，即通过控制先天免疫反应，以及积极的， 诱导病毒支持环境。这个子项目的目标是识别那些激酶控制的 支持黄病毒生命周期的宿主细胞途径。我们的初步数据显示， 蛋白酪氨酸激酶抑制剂阻止黄病毒进入，而MAP-激酶抑制剂干扰 复制的此外，我们先前证明Src激酶抑制剂阻断黄病毒的排出， 这是c-yes激酶对该步骤的要求。鉴定特异性抑制剂和特异性激酶 控制病毒生命周期的每一步，我们将使用化学基因组学方法。我们将使用基于细胞的， 高含量筛选激酶导向的小分子文库以鉴定激酶抑制剂 干扰登革病毒（DENV）生命周期的不同阶段。在二级屏幕中，我们将选择 具有高选择性指数、宽细胞类型特异性和抗其它黄病毒活性的化合物 (WNV，JEV，YFV）。活性化合物可能会抑制几种相关的激酶，从而最大限度地减少 信号通路的冗余和组织特异性表达。给定的靶向激酶 将通过结合计算、功能基因组学和生物化学方法来鉴定化合物。 基于这些结果，我们将生成控制黄病毒感染的激酶网络模型， 通过磷酸化蛋白质组学和靶向基因敲除实验检验这些模型。最后我们 将通过检查选择的激酶靶向抗病毒活性化合物的治疗潜力， 使用鼠模型西尼罗病毒感染在体内测定化合物。有效性、毒性和生物利用度 将激酶导向化合物的体内活性与TYT-1类似物（磺内酰胺硫脲）的体内活性进行比较。 在本项目的子项目3中生成。在这些全面研究结束时，我们预计 进一步了解了黄病毒与其宿主细胞的相互作用，确定了新的靶点， 抗病毒药物的发现，并表征了对黄病毒有活性的新型小分子抑制剂 在体外和体内。

Flaviviruses need and manipulate host cell pathways at every step of their infectious cycle. Since many host cell pathways are controlled by protein kinases, this class of host proteins is intimately involved in modulating flaviviral replication both negatively, ie. by controlling innate immune responses, as well as positively, by inducing a virus-supportive environment. The goal of this sub-project is to identify those kinase-controlled host cell pathways that support the flavivirus life cycle. Our preliminary data show that broad-spectrum protein tyrosine kinase inhibitors prevent flaviviral entry whereas MAP-kinase inhibitors interfere with replication. Moreover, we previously demonstrated that Src-kinase inhibitors block flaviviral egress due to a requirement of the c-yes kinase for this step. To identify both specific inhibitors and specific kinases controlling each step of the viral life cycle we will use a chemical genomics approach. We will employ cellbased, high content screening of kinase-directed small molecule libraries to identify kinase inhibitors interfering with different steps of the Dengue-virus (DENV) life cycle. In secondary screens we will select for compounds with a high selectivity index, broad cell type specificity and activity against other flaviviruses (WNV, JEV, YFV). Active compounds will likely inhibit several related kinases thus minimizing the problem of redundancy of signaling pathways and tissue specific expression. The kinases targeted by a given compound will be identified by combining computational, functional genomics and biochemical approaches. Based on these results we will generate models of kinase networks controlling flavivirus infection and examine thise models experimentally by phosphoproteomics and by targeted gene knockdown. Finally, we will examine the therapeutic potential of kinase-targeted antivirally active compounds by examining selected compounds in vivo using a murine model West-Nile Virus infection. The efficacy, toxicity and bio-availability of kinase-directed compounds will be compared with the in vivo activity of TYT-1 analogs (sultam thioureas) generated in sub-project 3 of this program. At the end of these comprehensive studies, we anticipate to have gained further insights into the interaction of flaviviruses with their host cell, identified novel targets for antviral drugs discovery, and characterized novel small molecule inhibitors that are active against flaviviruses in vitro and in vivo.