猪繁殖与呼吸综合征病毒非结构蛋白Nsp1β的SUMO化修饰及其生物学意义

The SUMOylation modification is an important post-translational protein modification which extensively exists in eukaryotic cells. Some viral proteins utilize the host's SUMOylation system to regulate their subcellular location, stability, and signaling transduction to promote viral multiplication, which is considered as viral immune evasion strategy. Porcine reproductive and respiratory syndrome (PRRS) is a relatively new viral infectious disease of the swine and has been become one of the most important diseases in countries with intensive swine industries. The nonstructural protein 1β (Nsp1β) encoded by PRRSV is an important nonstructural protein and plays critical role to regulate viral subgenomic transcription and to inhibit host's innate immunity. Our primary studies found that Nsp1β interacted with Ubc9, the SUMO-conjugating enzyme. We also demonstrated that Nsp1β was SUMOylated after co-expression SUMO1 protein. These results suggested that Nsp1β appears to be SUMOylated during PRRSV infection. However, the potent mechanism and its biological significance remain largely unknown. In this project, we will further investigate: (i) the SUMOylation modification of PRRSV Nsp1β by GST pulldown, in vivo and vitro sumoylation assays, and co-immunoprecipitation; (ii) the sumoylation site(s) in Nsp1β; (iii) the relationship between Nsp1β SUMOylation and its subcellular location and/or its ability to inhibit interferon production; (iv) the role of Nsp1β SUMOylation to PRRSV infection. Taken together, the present project is expected to reveal the potential immune evasion strategy of PRRSV from a new perspective, the post-translational protein modification.

SUMO化是一种广泛存在于真核细胞的蛋白质翻译后修饰，一些病毒蛋白利用宿主SUMO化修饰系统调节其亚细胞定位、稳定性和信号转导功能，促进病毒增殖，是病毒的一种免疫逃逸策略。猪繁殖与呼吸综合征（PRRS）是严重危害养猪业的病毒性传染病，PRRSV的非结构蛋白Nsp1β在病毒亚基因组转录和抑制宿主天然免疫方面发挥重要作用。我们的前期研究发现Nsp1β与SUMO化修饰结合酶Ubc9存在相互作用，与SUMO1共表达导致Nsp1β的SUMO化，但其被SUMO化修饰的机制及其生物学意义尚不清楚。本项目拟进一步从体内、体外两方面系统分析Nsp1β被SUMO化修饰的分子细节，确定SUMO化修饰位点，探讨SUMO化修饰对Nsp1β穿梭定位的影响，解析SUMO化修饰与Nsp1β抑制干扰素产生之间的关系，阐明Nsp1β的SUMO化修饰在病毒感染中的作用，从蛋白翻译后修饰这一新的角度揭示PRRSV的免疫抑制策略。

猪繁殖与呼吸综合征（PRRS）是危害全球养猪业最严重的病毒性传染病之一。PRRSV的免疫机制十分复杂，而且具有很强的免疫逃逸能力，这也是PRRS疫苗研制困难的重要原因。SUMO化是一种类泛素化修饰，也是一种广泛存在于真核细胞的蛋白质翻译后修饰。以前的研究发现，一些病毒编码蛋白可以利用宿主SUMO化修饰系统调节其亚细胞定位、稳定性和信号转导功能，促进病毒增殖，是病毒的一种免疫逃逸策略。本项目针对PRRSV编码的非结构蛋白nsp1β以及与之密切相关的nsp1α，对这两种非结构蛋白与宿主细胞的SUMO化修饰系统的相互作用进行了深入研究。利用在大肠杆菌中表达纯化的重组蛋白，制备了3株针对nsp1β的单克隆抗体和7株针对nsp1α的单克隆抗体；构建了表达nsp1α、nsp1β的重组慢病毒，绘制了超表达nsp1α、nsp1β后猪肺泡巨噬细胞的差异蛋白质组学图谱，从定量蛋白质组学角度证实nsp1α和nsp1β均具有抑制干扰素产生的特性；证实nsp1β能被SUMO1蛋白SUMO化，与SUMO1和Ubc9共表达能进一步促进nsp1β的SUMO化修饰，但在病毒感染条件下检测不到nsp1β的SUMO化修饰，我们推测在病毒感染过程中病毒编码的其他蛋白可能干扰了nsp1β的SUMO化修饰。在随后的研究中，我们发现nsp1α能阻碍细胞内SUMO化修饰水平，其N端的锌指结构域ZF1对阻碍细胞内SUMO化修饰的水平起着关键性的作用。同时还发现nsp1α阻碍IRF3的SUMO化修饰，促进IRF3的降解，参与RIG-I信号通路，从而抑制IFN-β产生；另外，我们还发现PRRSV nsp1α可以通过抑制线性化泛素复合体（LUBAC）介导的NEMO的线性化泛素修饰，从而拮抗LUBAC介导的NF-κB激活。上述研究结果从翻译后修饰的角度揭示了PRRSV的免疫调节新机制，为阐明PRRSV的致病与免疫机制奠定了基础。项目执行期内，先后在J Virol、Front Cell Infect Microbiol、J Proteomics、Proteomics等国际期刊上发表SCI论文5篇，获得国家发明专利1项，培养博士研究生4名、硕士研究生5名。