家蚕cGAMP- BmSTING抗病毒免疫信号通路研究

Disease caused by viral infection in silkworm, Bombyx mori results in serious loss in sericulture, therefore antiviral immunity has received much attention in silkworm research. cGAMP-STING signaling pathway has recently been demonstrated to be essential for viral DNA-mediated immune response and become one of the focus in mammalian innate immune research. However, whether this signaling pathway is involved in antiviral immune response in insect is unknown. Based on the evolutionary conservation between mammalian and insect innate immune system, we will first verify the presence of cGAMP produced by viral infected silkworm cells, identify BmSTING and its expression profiles as well as its ability to bind cGAMP. Then we will over-express BmSTING or reduce its expression level in both cells and in silkworm larvae to examine its role in antiviral immune response by measuring viral DNA replication, viral titer or larvae mortality after viral infection. In order to discover silkworm cytokines regulated by cGAMP-BmSTING pathway, we will identify molecules with antiviral activity secreted by cGAMP-stimulated cells from fractionated cell culture medium by combining with the transcriptome analysis. Lastly, we plan to interpret how BmSTING activates downstream transcriptional factors, such as BmRelish by identifying BmSTING-interacting molecules. Overall, this project aims to evaluate the antiviral immunity of cGAMP-BmSTING signaling pathway in silkworm cells and larvae, identify cytokines that can enhance antiviral immunity in naïve cells, and elucidate the mechanism of activating downstream transcriptional factors. We believe the results will improve our understanding of silkworm antiviral immune signaling pathway, and provide a reference for designing viral-resistance strategies.

家蚕病毒病是导致蚕业重大损失的原因之一，对家蚕抗病毒免疫机制的研究备受关注。cGAMP-STING信号通路近年来被证明在针对病毒DNA引起的免疫应答中起关键作用，因此在哺乳动物的先天免疫研究中备受关注，但在昆虫领域中尚无涉及。根据先天免疫系统的保守性，本项目将首先确定家蚕细胞在病毒感染后是否产生cGAMP，鉴定家蚕BmSTING并对其表达特征、结合cGAMP的能力予以检测。在细胞和个体水平上超表达BmSTING或降低BmSTING的表达水平，来检测cGAMP-BmSTING信号通路的开启是否有助于抗病毒免疫。通过对经cGAMP刺激后的细胞培养基上清液分级分离和质谱鉴定细胞所产生的具有抗病毒活性的分子，并结合转录组分析，筛选cGAMP-BmSTING信号通路调控的“细胞因子”，并验证其抗病毒活性。通过鉴定与BmSTING具有相互作用的分子，解析其激活BmRelish等转录因子的方式。

cGAMP-STING信号通路近年来被证明在哺乳动物抗病毒免疫应答中起关键作用，但在昆虫领域中尚无涉及。本项目首先鉴定了家蚕BmSTING并确定其表达特征，通过质谱鉴定了病毒感染的家蚕细胞产生cGAMP并确定了其随感染进程的水平变化。cGAMP刺激诱导抗病毒因子的产生，且BmSTING的亚细胞定位由细胞质弥散分布向核周点状聚集。利用dsRNA或CRISPRi降低BmSTING的表达水平，导致BmNPV感染后细胞中病毒DNA和BmNPV-GFP阳性细胞数均高于对照；而过表达BmSTING则结果相反。dsRNA注射降低家蚕幼虫体内BmSTING的表达，BmNPV感染后家蚕死亡率和病毒DNA的水平均高于对照。对比转染dsRFP和dsSTING的细胞经cGAMP诱导后的转录组测序结果，筛选到受cGAMP-BmSTING信号通路调控的分子并对其抗病毒活性予以了检测。分析候选分子启动子上的转录调控元件，推测其依赖转录因子BmRelish。cGAMP诱导、BmNPV感染或BmSTING过表达均导致BmRelish活化形式BmRelishact的产生并转运到细胞核内；在细胞中过表达BmRelishact，BmNPV感染后胞内病毒DNA的相对水平、BmNPV-GFP阳性细胞数均低于对照。过表达BmRelishact的细胞上清培养液具有抗病毒活性，通过质谱对其多肽成分进行了分析，并结合转录组分析，筛选了cGAMP-BmSTING信号通路调控的抗病毒因子。鉴定了与BmSTING互作蛋白BmCasp8L，该蛋白抑制了BmRelish的活化，但cGAMP刺激解除了抑制。BmCasp8L具有形成淀粉样蛋白聚集体的能力，通过与BmDREDD、BmFADD形成蛋白纤维聚合物抑制BmDREDD的活化，导致后者无法切割活化BmRelish。由此推测昆虫STING可能形成类似的信号复合体，通过实验验证了dSTING也具有形成淀粉样蛋白聚集体的能力，在胞内以多聚体形式存在，且RHIM基序是其形成聚合体及其与下游信号分子IMD相互作用的关键序列。本研究解析了cGAMP-BmSTING抗病毒免疫信号通路，并提出了STING等信号分子通过各自的特征基序/结构域组装成信号复合体的信号传递机制。