猪伪狂犬病毒变异株EP0蛋白拮抗干扰素通路介导免疫逃逸的机制

Pseudorabies virus (PRV), one of the most serious etiological agents, causes large economic losses for the swine industry. Immune escape is an important reason for its difficult eradication. PRV early protein EP0 is associated with pathogenicity and involved in the activation of viral gene transcription and reactivation from the latency. Previous studies showed that EP0 was important for PRV to antagonize the interferon (IFN) antiviral response, but the molecular mechanism is not clear so far. Viruses usually escape host immunity through degrading the innate immune molecules by interacting with them. So we speculate that PRV EP0 may interfere with IFN response through interacting with IFN signaling protein(s). To confirm this hypothesis, further studies will focus on screening the IFN signaling protein(s) which can be down-regulated by EP0 using CRISPR-Cas9 and confocal immunofluorescence assay, followed by identifying the interaction between EP0 and target protein(s) as well as the downregulation pathway and critical amino acid site(s) of EP0 through co-immunoprecipitation and gene site(s) mutagenesis. Furthermore, the effects of the interaction between EP0 and target protein(s) on viral replication and pathogenicity will be evaluated. These studies will not only provide novel insights into the mechanism of PRV immune escape and pathogenesis from the perspective of innate immunity, but also reveal new targets for anti-PRV medicine.

猪伪狂犬病毒（PRV）是严重危害养猪业的重要病原，免疫逃逸是其难以被根除的主要原因。PRV早期蛋白EP0参与病毒基因的转录及潜伏期的激活，与病毒致病性密切相关。前期研究发现EP0在PRV拮抗干扰素抗病毒应答中发挥重要作用，但确切机制尚未阐明。病毒通常利用与宿主先天性免疫分子相互作用将其降解的方式进行免疫逃逸，因此我们推测PRV EP0可能通过与干扰素通路相关蛋白的相互作用来拮抗宿主的干扰素应答。为证实这一假说，我们将通过CRISPR-Cas9、激光共聚焦等技术筛选能被EP0下调的干扰素相关分子（靶蛋白），并采用免疫共沉淀、基因定点突变等方法分析EP0与靶蛋白之间的相互作用及其下调靶蛋白表达的途径与关键氨基酸位点，进一步通过动物实验阐明EP0与靶蛋白的相互作用对PRV增殖与致病力的影响。本课题将从宿主先天性免疫的视角为PRV免疫逃逸与致病机制研究提供线索，也为抗PRV药物的研发提供新的靶点。

猪伪狂犬病毒（PRV）是严重危害养猪业的重要病原，免疫逃逸是其难以被根除的重要原因。PRV早期蛋白EP0参与病毒基因的转录及潜伏期的激活，与病毒的致病性密切相关。本研究制备了EP0蛋白的多克隆抗体和单克隆抗体，并通过CRISPR-Cas9技术构建PRV EP0基因缺失株，分析其与野生型的RPV的蛋白质组学表达差异，筛选出Sp100、Sp110、Spin1及PML等差异表达的宿主蛋白。然后，利用Western blot证实EP0能够明显下调Sp100、Sp110及Spin1的蛋白表达水平；利用免疫共沉淀（Co-IP）与激光共聚焦实验，证实PRV EP0能够与Sp100及Spin1蛋白发生相互作用。进一步，利用siRNA干扰技术干扰细胞中Spin1的表达，结果显示，与对照组相比，在感染后12 h，干扰Spin1能够显著促进PRV在PK-15细胞中的增殖。综上所述，本研究筛选出了Sp100、Sp110及Spin1等能够被PRV EP0下调的宿主抗病毒蛋白，而且PRV EP0通过与这些蛋白发生相互作用下调其蛋白表达水平，进而拮抗这些分子的先天抗病毒作用，阐明了PRV拮抗宿主抗病毒因子的一种新机制，为揭示PRV的致病机理以及新型药物的研发提供了理论参考。.