猪繁殖与呼吸综合征病毒非结构蛋白nsp2TF和nsp2N抑制Ⅰ型干扰素表达的分子机制研究

Modulation of host innate immune responses plays a key role in PRRSV immune evasion, persistent infection in naturally infected pigs, and current modified live vaccines lacking enough protection against heterologous virus challenge. The mechanism of PRRSV suppressing host innate immune responses is still not fully understood, although tremendous efforts have been made to study that. In our previous studies, we found two novel PRRSV nonstructural proteins, nsp2TF and nsp2N, which are important for virus replication but not essential for virus viability. These two proteins showed an inhibitory effect on the type I interferon (IFN) production. Furthermore, in comparison with the wild type PRRSV, nsp2TF/nsp2N deficient recombinant viruses expedited the IFN-α response, increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Therefore, nsp2TF and nsp2N appear to play an important role in the mechanism of PRRSV immune evasion. Currently, the mechanism of nsp2TF and nsp2N suppressing host type I IFN production is unknown. Based on these results, we proposed to study the mechanism of nsp2TF and nsp2N suppressing host type I IFN response focusing on 3 specific aims. In Aim 1, we will identify the molecule(s) in the type I IFN production signaling pathway targeted by nsp2TF and nsp2N and the detailed mechanism. In Aim 2, we will identify the key region(s) and residue(s) of nsp2TF and nsp2N which are critical for their ability to suppress type I IFN production. In Aim 3, using PRRSV reverse genetics, we will introduce mutations at the identified key elements to rescue recombinant viruses with improved ability in stimulation of host immune responses. The data generated in this study will provide important information on virus-host interactions and the basis for the development of next-generation vaccine.

调控宿主天然免疫反应是猪繁殖与呼吸综合征病毒（PRRSV）逃避宿主抗病毒免疫反应，形成持续性感染以及当前疫苗缺乏有效交叉保护的重要因素，其分子机制尚未完全阐明。申请者在前期工作中发现了两个新的PRRSV非结构蛋白（nsp2TF和nsp2N），证实它们不是病毒复制的必需蛋白、能够显著抑制I型干扰素表达。敲除nsp2TF和nsp2N的表达能够显著提高突变病毒刺激I型干扰素表达，干扰素刺激基因表达以及细胞免疫反应的能力，可见nsp2TF和nsp2N抑制I型干扰素表达对PRRSV逃逸宿主免疫反应至关重要。本项目拟在此基础上，采用双荧光素酶报告系统、免疫共沉淀、共聚焦显微镜及反向遗传操作系统等方法探究nsp2TF和nsp2N抑制I型干扰素表达的分子机制，鉴定介导它们免疫抑制功能的关键蛋白序列或氨基酸位点，对阐明PRRSV与宿主的互作机制有重要意义，为研发新一代疫苗奠定基础。

调控宿主天然免疫反应是猪繁殖与呼吸综合征病毒（PRRSV）逃避宿主抗病毒免疫反应，形成持续性感染以及当前疫苗缺乏有效交叉保护的重要因素，其分子机制尚未完全阐明。本研究以HP-PRRSV TA-12毒株为研究对象，解析了nsp2，nsp2TF和nsp2N抑制I型干扰素表达的分子机制。本研究的主要成果包括：鉴定了IFNβ表达信号通路中被nsp2相关蛋白靶向抑制的宿主因子为TBK1/IKKε；PLP2的蛋白酶结构域去除TBK1/IKKε的K63连接的泛素化修饰，从而抑制它们的活化及下游信号转导；nsp2相关蛋白的半胱氨酸蛋白酶活性位点是它们抑制IFN-β表达的关键氨基酸位点；发现PRRSV nsp2相关蛋白抑制IFNβ表达具有毒株差异性；敲除nsp2TF和nsp2N拯救的突变病毒KO2具有显著提高的刺激I型干扰素和干扰素刺激基因表达的能力。研究结果对深入解析HP-PRRSV的免疫抑制机制具有重要的科学意义，为疫苗研发和改造提供了理论基础。