Mechanistic Insights into flavivirus NS5-mediated STAT2 Suppression

黄病毒 NS5 介导的 STAT2 抑制的机制见解

• 批准号: 10371157
• 负责人: Rong Hai
• 金额: $ 38.88万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10371157
• 关键词: Affect; Antiviral Agents; Attenuated Vaccines; Binding; Binding Proteins; Biochemical; Cells; Complex; Cryoelectron Microscopy; Crystallization; Data; Dengue Virus; Development; Epidemic; Evaluation; Family; Flavivirus; Flavivirus Infections; Gene Expression; Goals; Host Defense; Human; In Vitro; Infection; Integration Host Factors; Interferons; Knowledge; Link; Mediating; Molecular; Mutation; Nonstructural Protein; Pathogenicity; Pathway interactions; Physiological; Process; Proteins; Public Health; RNA; Role; STAT2 gene; Signal Pathway; Signal Transduction; Species Specificity; Specificity; Stat2 protein; Techniques; Testing; Therapeutic; Vaccines; Viral; Viral Pathogenesis; Virus; Virus Diseases; X-Ray Crystallography; Zika Virus; antagonist; base; global health; in vivo; inhibitor; insight; interferon-stimulated gene factor 3; member; molecular recognition; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; novel therapeutics; prevent; prophylactic; protein degradation; response; ubiquitin-protein ligase; vector-borne; virology

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.

黄病毒家族由90多种媒介传播的单链RNA病毒组成，包括登革病毒(DENV)和寨卡病毒(ZIKV)，它们在人类中引起重大流行，并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。要确定感染，黄病毒需要克服1型干扰素(干扰素-1)诱导的抗病毒状态，这是宿主防御的第一道防线。在这方面，黄病毒编码了几种拮抗剂来抑制干扰素反应。例如，DENV、ZIKV和其他一些黄病毒的非结构NS5蛋白已被证明是干扰素信号的有效抑制因子，针对干扰素信号通路的不同步骤。和DENV一样，ZIKV NS5蛋白与人类信号转导和转录激活因子2(HSTAT2)蛋白结合并触发其蛋白酶体降解，尽管使用了不同的下游机制。为了阐明黄病毒NS5介导的hSTAT2抑制的机制基础，我们建议提供对ZIKV NS5-hSTAT2和DENV NS5-hSTAT2复合体的结构洞察，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降解和干扰素信号抑制中的后果(S)的询问。为此，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。在目标1中，我们将通过X射线结晶学和冷冻电子显微镜建立ZIKV-hSTAT2相互作用的结构基础，并通过突变和体外下拉分析验证我们的观察结果。在目标2中，我们将在细胞水平上研究ZIKV NS5-hSTAT2的相互作用，并通过评估ZIKV NS5对hSTAT2降解、干扰素应答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特异性ZIKV NS5-hSTAT2的相互作用提供关键的结构和功能方面的见解，从而在黄病毒NS5蛋白、hSTAT2降解、干扰素反应抑制和病毒感染之间建立机制联系。拟议研究的结果最终将有利于新型抗病毒药物和抗黄病毒感染的活疫苗的开发。