蓝舌病病毒抑制JAK-STAT通路内I型干扰素信号传导机制研究

Bluetongue virus (BTV), an arthropod-borne pathogen, infects ruminants such as cattle and sheep, and causes an often fatal, hemorrhagic disease including bluetongue (BT) in ruminants. In addition to reduced economic productivity of the livestock, BTV leads to direct economic losses due to the death of the infected livestock. As global climate changing, BT has showed a tendency of continuing spread around the world, threatening seriously animal husbandry production. Studies have shown that BTV inhibits type I interferon signaling and partially antagonizes the antiviral activity of the host innate immune system by targeting Janus tyrosine kinase (JAK)-signal transducer and activator of transcription protein (STAT) pathway. This process requires the contribution of two or more viral proteins. But so far, both these viral proteins that exert synergistically antagonism and their mechanisms of action are still unclear. In this study, mass spectrometry and luciferase reporter gene assays were firstly used to determine the viral proteins that synergistically antagonize type I interferon. Secondly, molecular and biochemical methods were performed to investigate the interrelationship between these viral proteins and host proteins in the JAK-STAT pathway and the mechanisms of these viral proteins antagonizing type I interferon signaling. Further understanding of the mechanisms for BTV viral proteins interfering with the host innate immune response through JAK-STAT pathway will advance the scientific basis for the development of new type of vaccines and provide better measures for the prevention and control of BT.

蓝舌病病毒(BTV)是一种由节肢动物传播的病原体，感染反刍动物，引起蓝舌病(BT)等致命的出血性疾病。除导致牲畜减产外，BTV还导致被感染动物死亡，造成直接经济损失。由于全球气候变暖，蓝舌病在全世界范围内呈不断扩散趋势，严重威胁畜牧业生产。研究发现BTV通过JAK-STAT通路抑制I型干扰素信号传导，并部分拮抗宿主天然免疫系统的抗病毒作用；该过程需要多个病毒蛋白协同发挥作用。但是，我们尚不清楚这些发挥拮抗作用的病毒蛋白及其作用机制。本研究首先通过蛋白质谱技术和荧光素酶报告系统确定协同拮抗I型干扰素的BTV蛋白。其次，通过分子生物学和生物化学手段确定这些病毒蛋白与JAK-STAT信号通路内宿主蛋白间的互作关系，揭示BTV拮抗I型干扰素信号传导的机制，并进一步丰富对BTV蛋白通过JAK-STAT通路干扰宿主天然免疫应答的理解，为开发新型疫苗提供科学依据，为预防和控制BT提供更好的措施。

BTV能够感染牛、羊和鹿等家养及野生反刍动物，导致被感染动物发生出血性死亡，给全球畜牧业生产带来巨大经济损失。病毒一旦入侵宿主，必然会受到宿主天然免疫系统的攻击，BTV也不例外。然而，研究发现BTV能够拮抗IFN-I信号在JAK-STAT通路内的传导进而有利于自身繁殖，但是该过程涉及的具体机制尚不完全清楚。本研究首先利用免疫印迹、免疫荧光和免疫共沉淀技术对JAK-STAT信号通路内重要转录因子STAT1和STAT2的表达、磷酸化、二聚化和核转移水平进行了检测；其次，通过免疫共沉淀-质谱技术确定与STAT1相互作用的BTV-1病毒蛋白；再次，利用RT-qPCR明确过表达相关病毒蛋白对JAK-STAT通路下游基因转录水平的影响；最后，通过免疫共沉淀、GST pull-down和邻位连接技术确定BTV-1病毒蛋白与STAT1相互作用关系及相互作用的结构域。研究结果表明，BTV-1对STAT1和STAT2的表达水平以及STAT2磷酸化水平影响较小，主要通过影响JAK-STAT通路内STAT1的磷酸化水平而降低其二聚化和核转移水平；BTV-1利用其非结构蛋白NS3和NS4与STAT1相互作用；而过表达BTV-1的NS3和NS4能够导致JAK-STAT通路下游ISGs转录水平降低；BTV-1的NS3和NS4均与STAT1直接相互作用，并均结合于STAT1的SH2结构域。通过对BTV-1拮抗IFN-I信号在JAK-STAT通路内传导的机制进行研究，有助于系统而全面地揭示BTV免疫逃逸现象背后的本质，进而深入理解BTV的感染和致病机制，为开发防治BTV的新技术和新方法以及研制更加有效和安全的BTV疫苗提供科学数据。