EV71编码的2A蛋白酶下调干扰素受体降解机理的研究

Since 2008, the repeated large outbreaks of enterovirus 71 (EV71) has caused to thousands of deaths and more neurological complications of children in China annually. It was not understood why the conventional interferons take limited anti-EV71 effects on both animal studies and clinical settings. Recently, for the first time, we have shown that EV71 disrupts interferon signaling by reducing its receptor IFNAR1 level in host cells. More importantly, we have also shown that viral protease 2A plays an important role in the process. In this study, we will elucidate the relationship between IFNR1 and ubiquitin-mediated protein degradation. We will investigate if IFNAR1 was ubiquitinated, and the mechanism of internalization and degradation of IFNAR1 promoted by EV71. We will use multiple approaches, including Transcriptional regulation assay, in vitro translation assay, Fluorescent-based receptor internalization assay, Protein degradation assay, Pull down assay, Co-immunoprecipiation assay and MALDI-TOF/TOF tandem mass spectrometer assays to demonstrate the molecular mechanisms of EV71 used to reduce the IFNAR1 levels of host cells as well as the corresponding pathogenesis. Furthermore, we will also identify the targets (substrate or other interacting proteins) of 2A and further dissect the function of bingding protein of protease 2A. The output of this creative study will not only reveal the molecular mechanism of EV71 for reducing the IFNAR1 level, but also demonstrate potential drug target and lay down the fundamental basis for antiviral therapies.

自2008年以来，EV71病毒在我国多次流行，已导致上千儿童死亡及中枢神经系统疾病后遗症。干扰素对EV71感染疗效甚微,其机制尚不清楚。本课题组首次证实EV71阻断干扰素信号通路以消除其抗病毒效果。特别是EV71感染导致干扰素受体水平下降，与其在细胞膜上的聚集而可能导致的降解有关，而病毒2A蛋白酶在此过程中起着关键作用。本课题将通过免疫共沉淀方法来揭示干扰素受体（简称IFNAR1）与泛素蛋白的关系，检测IFNAR1是否被泛素化以及观察IFNAR1的内在化，研究EV71病毒促进IFNAR1内在化和降解的机制；通过Pull down、免疫共沉淀和质谱分析等技术揭示2A蛋白酶的靶点，寻找和鉴定与病毒蛋白2A结合并共同调控干扰素受体的靶分子，研究病毒蛋白2A的结合蛋白的功能。本研究将揭示EV71消除干扰素疗效的原因、揭示其致病机理和发现新的药物靶点，为设计新的抗病毒药物提供思路和科学依据。

最近71型肠病毒（EV71）感染了100多万名儿童，造成约1000人死亡。 迄今为止，还没有使用有效的疫苗或疗法来预防或治疗EV71感染。几十年来，干扰素已成功用于治疗乙肝和丙肝病毒感染的患者，然而干扰素在治疗EV71感染方面尚未有效。. 本研究小组已经研究了EV71感染的机制和治疗策略，并且已经确定了作为干扰素信号传导拮抗剂的基本病毒成分。已经发现EV71通过下调干扰素α受体1（IFNAR1）拮抗I型干扰素信号传导。在这项研究中，我们将确定EV71的宿主蛋白质目标，并探讨EV71用于下调IFNAR1的机制。（1）我们发现2Apro与EV71在干扰信号转导中发挥类似的作用——下调IFNAR1，从而抑制STAT1和STAT2的TYK2和JAK1的磷酸化；（2）证实2Apro不影响IFNAR1的转录，IFNAR1 mRNA的稳定性和IFNAR1的翻译；（3）我们发现2Apro结合IFNAR2，通过内化分选促进IFNAR1的降解；（4）以重组2Apro和2AC110A突变体作为诱饵，我们拉下了一些2Apro相互作用蛋白。在MOSS TOF / TOF MS分析后，我们成功地回收了16个蛋白质ID。通过生物信息学分析，我们选择了三种蛋白HSc70，hnRNP L和RAP进行进一步的生物学研究; （5）证明HSc70与2Apro相互作用，HSc70的敲低通过增加IFNAR1的表达来激活IFN信号来抑制EV71的复制和繁殖，并且HSc70抑制剂将成为抗EV71感染的潜在抗病毒剂。