Mechanistic Insights into flavivirus NS5-mediated STAT2 Suppression

黄病毒科由90多种媒介传播的单链rna病毒组成，包括登革热病毒（DENV）和寨卡病毒（ZIKV），它们在人类中引起重大流行病，对全球公共卫生构成严重威胁。目前还没有疫苗或抗病毒药物来预防或治疗由DENV、ZIKV和其他一些黄病毒引起的感染。为了建立感染，黄病毒需要克服1型干扰素（IFN-1）诱导的抗病毒状态，这是宿主的第一道防线。在这方面，黄病毒已经编码了几种拮抗剂来抑制IFN的反应。例如，DENV、ZIKV和其他一些黄病毒的非结构NS5蛋白已被证明是IFN信号通路的有效抑制因子，靶向IFN信号通路的不同步骤。与DENV一样，ZIKV NS5蛋白结合人信号换能器和转录激活因子2 （hSTAT2）蛋白并触发其蛋白酶体降解，尽管使用不同的下游机制。为了阐明黄病毒ns5介导hSTAT2抑制的机制基础，我们建议对ZIKV NS5-hSTAT2和DENV NS5-hSTAT2复合物进行结构分析，这反过来将指导对黄病毒NS5-hSTAT2相互作用在蛋白酶体介导的hSTAT2降解和IFN信号抑制中的后果的研究。为了实现这一目标，我们将使用结构、生化、分子、细胞和病毒学方法来研究ZIKV NS5-hSTAT2和DENV NS5-hSTAT2相互作用的结构基础及其功能后果。在Aim 1中，我们将通过x射线晶体学和低温电镜建立ZIKV-hSTAT2相互作用的结构基础，并通过突变和体外拉下分析验证我们的观察结果。在Aim 2中，我们将在细胞水平上研究ZIKV NS5-hSTAT2相互作用，并通过评估ZIKV NS5对hSTAT2降解、IFN反应和病毒感染的突变效应来研究ZIKV NS5-hSTAT2相互作用的功能后果。这些研究结果将为病毒和物种特异性ZIKV NS5-hSTAT2相互作用提供关键的结构和功能见解，从而建立黄病毒NS5蛋白、hSTAT2降解、IFN反应抑制和病毒感染之间的机制联系。拟议研究的结果将最终有利于开发抗黄病毒感染的新型抗病毒药物和活疫苗。

The family of flavivirus consists of over 90 vector-borne, single-stranded RNA-containing viruses, including Dengue virus (DENV) and Zika virus (ZIKV), which cause major epidemics among humans and pose a serious threat to global public health. No vaccines or antivirals exist to prevent or treat infections caused by DENV, ZIKV, and some other flaviviruses. To establish infection, flaviviruses need to overcome the antiviral state induced by type 1 interferon (IFN-1), the first line of host defense. In this regard, flaviviruses have encoded several antagonists to suppress IFN responses. For example, the nonstructural NS5 proteins of DENV, ZIKV, and some other flaviviruses have been shown to be potent suppressor of IFN signaling, targeting different steps of the IFN signaling pathway. Like DENV, ZIKV NS5 protein bind human signal transducer and activator of transcription 2 (hSTAT2) protein and trigger its proteasomal degradation, albeit using different downstream mechanisms. To elucidate the mechanistic basis of flavivirus NS5-mediated hSTAT2 suppression, we propose to provide structural insight into the ZIKV NS5-hSTAT2 and DENV NS5-hSTAT2 complexes, which, in turn, will guide interrogation of the consequence(s) of the flavivirus NS5-hSTAT2 interactions in proteasome-mediated degradation of hSTAT2 and suppression of IFN signaling. Toward this goal, we will use structural, biochemical, molecular, cellular and virology approaches to investigate the structural basis of the ZIKV NS5-hSTAT2 and DENV NS5-hSTAT2 interactions and their functional consequence. In Aim 1, we will establish the structural basis of the ZIKV-hSTAT2 interaction by using X-ray crystallography and cryo-electron microscopy and validate our observations with mutational and in vitro pull-down analyses. In Aim 2, we will examine the ZIKV NS5-hSTAT2 interaction at a cellular level and investigate the functional consequence of the ZIKV NS5-hSTAT2 interaction through evaluation of the mutational effects of ZIKV NS5 on hSTAT2 degradation, IFN response and viral infection. The results of these studies will provide critical structural and functional insights into the virus- and species-specific ZIKV NS5-hSTAT2 interaction, thereby establishing a mechanistic link between flavivirus NS5 proteins, hSTAT2 degradation, suppression of the IFN response and viral infection. Results from the proposed studies will ultimately benefit development of novel antivirals and live vaccines against flaviviruses infection.