猪繁殖与呼吸综合征病毒N蛋白SUMO化修饰对病毒复制及致病性的影响

SUMOylation, which targets substrates with small ubiquitin-like modifier (SUMO), has emerged as an essential theme of posttranslational modification in regulating a wide range of cellular processes. Recent advances in this field have revealed that the SUMOylation of virus proteins play an important role in suppressing the host innate immune responses, viral immune evasion and virus replication. Porcine reproductive and respiratory syndrome virus (PRRSV), one of the most serious etiological agent in pig breeding worldwide. PRRSV contains ten open reading frames (ORFs) and encodes at least eight structural proteins. Among them, the highly immunogenic nucleocapsid protein (N), a significant integral component of virus particles, plays a crucial role in RNA synthesis and the assembly of infectious particles. Our preliminary study indicated that PRRSV N protein interacted with Ubc9, the E2 SUMO conjugating enzyme and it also confirmed the N protein was SUMOylized in its transfected cells and PRRSV infected ones. However, the mechanism and biological significances of PRRSV N SUMOylation remain to be answered. Further studies will focus on identifying the SUMOylation site(s) through co-immunoprecipitation, GST pull down, confocal immunofluorescence assay and site(s) mutagenesis, followed by evaluating the effects of PRRSV N SUMOylation on the viral growth efficiency both in vitro and in vivo, as well as the pathogenicity for piglets via infectious clone technology. These studies may not only provide some insights into the mechanism of PRRSV pathogenesis and immunosuppression from a new perspective, it may also reveal new targets for anti-PRRSV.

SUMO是一类重要的类泛素蛋白，广泛参与生物体生命活动。病毒蛋白的SUMO化修饰在抑制宿主先天性免疫、参与病毒免疫逃逸及复制中发挥重要作用。猪繁殖与呼吸综合征病毒（PRRSV）是严重危害养猪生产的重要病原，其核衣壳蛋白（N蛋白）是病毒粒子重要的内部结构蛋白及免疫原性蛋白，参与病毒RNA合成及感染性病毒颗粒装配，N蛋白入核与PRRSV致病性密切相关。我们前期研究发现PRRSV N蛋白可与SUMO结合酶Ubc9相互作用，进而发生SUMO化修饰，但该过程的具体机制及生物学意义尚不明确。本项目拟通过免疫共沉淀、pull down等技术确定N蛋白SUMO化修饰位点，利用感染性克隆平台分析SUMO化修饰对PRRSV体外复制能力的影响，动物实验确证N蛋白SUMO化修饰对PRRSV增殖力及致病性的影响，从蛋白翻译后修饰的新视角为PRRSV致病机制和免疫抑制研究提供线索，也为抗PRRSV药物研发提供新靶点。

猪繁殖与呼吸综合征病毒（PRRSV）是危害世界养猪生产的重要病原，并造成巨大的经济损失。其核衣壳蛋白（N蛋白）是病毒粒子重要的内部结构蛋白及免疫原性蛋白，参与病毒RNA合成及感染性病毒颗粒装配，且N蛋白核定位（Nuclear Localization Signal，NLS）与PRRSV致病性密切相关。SUMO是一类重要的类泛素蛋白，广泛参与生物体生命活动，病毒蛋白的SUMO化修饰在抑制宿主先天性免疫、参与病毒免疫逃逸及复制中发挥重要作用。本论文围绕PRRSV N蛋白SUMO化修饰对病毒体外复制、N蛋白亚细胞定位和抑制β干扰素产生的影响开展研究，旨在从蛋白翻译后修饰的新视角为PRRSV致病机制研究提供线索，也为抗PRRSV药物研发提供新靶点。.本研究验证了PRRSV Nsp1β、Nsp4、Nsp9、Nsp10及N蛋白与宿主细胞蛋白Ubc9存在相互作用，证实了PRRSV N蛋白存在SUMO化修饰现象，并确定赖氨酸对N蛋白SUMO化修饰是冗余的。通过构建N蛋白赖氨酸突变体及拯救其突变病毒，证实N蛋白赖氨酸突变体可影响IFN-β和IRFs启动子活性及改变N蛋白的亚细胞定位；除第7,28,39,52位赖氨酸外，N蛋白其余赖氨酸与病毒的体外增殖能力密切相关，且与诱导IFN-β水平有关。