Flavivirus NS3-mediated Innate Immune Escape

项目摘要 由蚊子传播的黄病毒，包括登革热（DV）和西尼罗河病毒（WNV） 寨卡病毒（ZIKV）是寨卡病毒的一种，是一种全球性的流行病。 关心尽管由这些病毒感染导致的高发病率和死亡率 病原体，目前没有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。 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力、菌株毒力、宿主遗传学和病毒-宿主相互作用。 在调节发病机制的病毒-宿主相互作用中，病毒介导的对 先天免疫信号通路具有关键作用。然而， 哪些黄病毒逃避宿主先天免疫还没有很好的表征。 这项拟议的研究建立在Gack实验室最近的一项发现基础上，该发现表明， DV与细胞靶向运输蛋白14-3-3ε相互作用以阻断细胞质到细胞质的转运。 病毒RNA传感器RIG-I的线粒体易位，从而抑制抗病毒信号传导 和I型干扰素（IFN）诱导。我们已经确定了DV NS 3中14-3-3ε的精确基序 相互作用，一个四个氨基酸的磷酸模拟64 RxEP 67基序，也产生了一个 重组突变体DV。该重组病毒编码一个14-3-3ε-结合缺陷突变体， NS 3蛋白在具有完整先天宿主的细胞中与野生型DV相比生长减弱 体外防御和激发强大的先天免疫和T细胞应答。WNV和ZIKV NS 3 蛋白质编码类似的磷酸化模拟基序，64 RLDP 67，我们的初步结果表明， WNV NS 3也靶向14-3-3ε以阻断RIG-I介导的先天免疫。 利用分子、生物化学和细胞生物学方法结合感染研究 用重组NS 3突变病毒，我们将精确详细地定义DV和WNV NS 3 抑制宿主IFN应答。本研究还将深入了解 ZIKV NS 3蛋白调节IFN介导的宿主防御反应（Aim 1）。最后我们将 确定14-3-3ε-NS 3相互作用与病毒发病机制的生理相关性， 使用体外细胞培养系统和小鼠逃避宿主先天性和适应性免疫 WNV感染模型（目的2）。我们的研究将提供一个分子的理解， DV，WNV和ZIKV的免疫逃逸机制，这可能指导合理设计 新的疫苗和抗病毒药

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.