Flavivirus NS3-mediated Innate Immune Escape

项目总结 由蚊子传播的黄病毒，包括登革热(DV)和西尼罗河病毒(WNV) 病毒以及最近出现的寨卡病毒(ZIKV)代表着重大的全球健康 担忧。尽管感染这些病毒导致的发病率和死亡率很高 病原体，目前还没有FDA批准的治疗方法。因此，迫切需要 更好地了解这些病毒的发病机制，以帮助设计疫苗和抗病毒药物。 人类病毒感染的疾病严重程度和发病机制取决于许多因素， 包括预先存在的免疫、菌株毒力、宿主遗传学和病毒与宿主的相互作用。 在调节致病的病毒-宿主相互作用中，病毒介导的抑制 先天免疫信号通路起着至关重要的作用。然而，精确的机制是通过 哪些黄病毒可以逃避宿主的天然免疫，目前还没有得到很好的描述。 这项拟议的研究建立在Gack实验室最近的一项发现基础上，即NS3蛋白 DV与线粒体靶向运输蛋白14-3-3ε相互作用，阻断胞浆到胞浆 病毒RNA传感器RIG-I的线粒体易位，从而抑制抗病毒信号 I型干扰素诱导。我们已经确定了DVNS3中14-3-3ε的确切基序 相互作用，一个四氨基酸的仿64RxEP67基序，并产生了一个 重组突变体DV。该重组DV编码14-3-3ε结合缺陷突变体 在具有完整天然宿主的细胞中，与野生型DV相比，NS3蛋白的生长减弱 并在体外诱导强大的先天免疫和T细胞反应。西尼罗河病毒和ZIKV NS3 我们的初步结果显示，蛋白质编码一个类似的模拟磷化基序64RLDP67 西尼罗河病毒NS3还以14-3-3ε为靶点，阻断RIG-I介导的天然免疫。 使用分子、生物化学和细胞生物学方法结合感染研究 对于重组NS3突变病毒，我们将精确详细地定义DV和WNV NS3是如何 抑制宿主的干扰素反应。这项研究也将深入了解(S) ZIKV NS3蛋白调节干扰素介导的宿主防御反应(目标1)。最后，我们会 确定14-3-3ε-NS3相互作用与病毒致病和 利用体外细胞培养系统和小鼠逃避宿主天然免疫和获得性免疫 西尼罗河病毒感染模型(目标2)。我们的研究将提供一个分子上的理解 DV、WNV和ZIKV的免疫逃逸机制，可指导合理设计 新的疫苗和抗病毒药物。

PROJECT SUMMARY Flaviviruses that are transmitted by mosquitoes, including dengue (DV) and West Nile (WNV) viruses as well as the recently emerging Zika virus (ZIKV), represent a significant global health concern. Despite the high morbidity and mortality resulting from infection by these viral pathogens, there are currently no FDA-approved therapies. Hence, there is a pressing need to understand better the pathogenesis of these viruses to aid the design of vaccines and antivirals. Disease severity and pathogenesis of viral infections in humans depend on many factors, including pre-existing immunity, strain virulence, host genetics and virus-host interactions. Among the virus-host interactions that modulate pathogenesis, virus-mediated suppression of innate immune signaling pathways has a critical role. However, the precise mechanisms by which flaviviruses evade host innate immunity are not well characterized. The proposed study builds on a recent discovery by the Gack laboratory that the NS3 protein of DV interacts with the mitochondrial-targeting trafficking protein 14-3-3ε to block the cytosol-to- mitochondria translocation of the viral RNA sensor RIG-I, thereby suppressing antiviral signaling and type I interferon (IFN) induction. We have identified the precise motif in DV NS3 for 14-3-3ε interaction, a four-amino-acid phosphomimetic 64RxEP67 motif, and also generated a recombinant mutant DV. This recombinant DV, encoding a 14-3-3ε-binding-deficient mutant NS3 protein, is growth-attenuated compared to wild-type DV in cells with intact innate host defense and elicits robust innate immune and T cell responses in vitro. WNV and ZIKV NS3 proteins encode a similar phosphomimetic motif, 64RLDP67, and our preliminary results show that WNV NS3 also targets 14-3-3ε to block RIG-I-mediated innate immunity. Using molecular, biochemical and cell biological approaches combined with infection studies with recombinant NS3 mutant viruses, we will define in precise detail how DV and WNV NS3 inhibits the host IFN response. This study also will yield insight into the mechanism(s) by which ZIKV NS3 protein modulates IFN-mediated host defense responses (Aim 1). Finally, we will determine the physiological relevance of the 14-3-3ε-NS3 interaction for viral pathogenesis and escape from host innate and adaptive immunity using in vitro cell culture systems and mouse models of WNV infection (Aim 2). Our studies will provide a molecular understanding of the immune escape mechanisms of DV, WNV and ZIKV, which may guide the rational design of new vaccines and antivirals.