家猪干扰素诱导表达信号通道RIG-I与病毒PRRSV 抑制的研究

In the last decade, the research on antiviral innate immune response has demonstrated that the initiation of IFN-β transcription is a key regulating step to promote secretion of type I IFN and RIG-I (Retinoic-acid-inducible gene-I) signaling pathway plays a major role in the detection of pathogens-associated molecular patterns (PAMPs) of viral RNA and activation of IFN-β production. Many viruses, however, have developed complicated strategies against innate defense, one of which is blocking RIG-I signaling cascade by suppressing its adaptor proteins. Porcine reproductive and respiratory syndrome (PRRS) is a major swine disease, causing significant economic losses for swine industry worldwide. Infection of the causative agent, PRSS virus, is resulted in a weak and atypical innate immune response, especially in lack of type I IFN. The aberrant host innate defense might not be caused by an inadequate recognition of the virus, but by modulation of the host antiviral signaling pathways with PRRSV, as many other RNA viruses. In the proposed project, the suppression of IFN-β gene transcription by blocking RIG-I signaling pathway in porcine cells will be investigated. Viral proteins of PRRSV are expressed in competent porcine cells individually to determine which protein can block the dsRNA-induced IFN-β expression through RIG-I pathway. The positive suppressor proteins are then expressed in porcine alveolar macrophage (PAM) to study their suppression in different porcine breeds. To further address the differential blocking of INF-β induction between porcine breeds, RIG-I and the signaling adaptor proteins of two breeds that are differentially susceptible to PRRSV, including Chinese Tibetan pigs and the one introduced from western countries, are cloned and sequenced. The amino acid polymorphic type of each signaling protein is then used to trace the breed-specific signaling protein suppressed by viral protein in the following experiments: (1) co-expression of signaling and viral proteins in competent PAM and detecting the activation of RIG-I pathway by dsRNA; (2) construction of porcine reporter gene to show the suppressing RIG-I pathway in different breeds while expression of viral protein; and (3) reconstitution of RIG-I pathway by adding gene-modified protein to replace the knockout signaling protein to estimate the pathway resistant to viral protein suppressing. Based on the information above, the signaling protein of porcine RIG-I pathway suppressed by PRRSV protein may be determined and a chimeric porcine RIG-I pathway resistant to viral protein suppressing may also be reconstituted by replacing the sensitive protein with the one of other porcine breeds or the one gene-modified. Therefore, the proposed research will help to understand the mechanism on suppression of IFN-β production through RIG-I pathway by PRRSV and provide an approach to overcome the suppression by reconstitution of porcine RIG-I pathway with non-susceptible signaling proteins.

近年来，对病毒RNA诱导宿主先天免疫应答研究发现，RIG-I信号通道是细胞识别病毒RNA、激活干扰素表达的重要路径，也是多种病毒调控宿主先天免疫应答的作用对象。本项目拟对猪繁殖与呼吸综合征病毒（PRRSV）抑制宿主RIG-I信号通道的问题进行研究。首先，对家猪RIG-I信号通道分子克隆和测序，研究它们在西方品系和藏猪中的遗传多样性。再将PRRSV蛋白分别表达，在敏感品系中鉴定出抑制RIG-I通道的病毒蛋白，测试对其它品系的抑制差异。通过比较RIG-I通道遗传多样性品系受病毒蛋白抑制的差异、表达或敲除通道分子、检测通道分子受抑制的效应等，发现和确定受病毒蛋白抑制的RIG-I通道分子。最后，用免受抑制的通道蛋白（来自不同品系或物种或经基因改造）和其它信号通道分子，重建抑制解除的RIG-I信号通道。以上研究将为阐明PRRSV抑制RIG-I信号通道机制、发现和创造动物抗病毒基因资源提供理论支持。

在本项目获准的一年研究时间内，课题组围绕家猪先天免疫信号通道蛋白的遗传多样性和表达水平对宿主PRRSV易感性的影响展开研究。主要进行了：1）藏猪先天免疫信号通道RIG-1蛋白分子的基因克隆、测序，与其它品系同类分子的序列比较分析；2）藏猪、约克猪肺泡巨噬细胞体外病毒感染试验，约克猪、荣昌猪PRRSV攻毒试验；3）藏猪和约克猪肺泡巨噬细胞数字基因表达谱（DGEs）建立及先天免疫相关基因表达分析。研究结果显示，藏猪先天免疫信号通道RIG-1主要分子的核酸和氨基酸序列都与西方品系存在明显差异，它们可作为分子标记区分PRRSV易感性差异个体，鉴定藏猪杂交后代分离群体中不同易感性个体的基因型，筛选病毒抗性或耐受性基因。肺泡巨噬细胞体外攻毒和活体攻毒试验显示，藏猪、约克、荣昌猪对PRRSV的易感性存在差异，如肺泡巨噬细胞攻毒的感染率和活体攻毒后宿主反应（病症、体温变化、失去体重、病毒繁殖等）。藏猪肺泡巨噬细胞数字基因表达谱分析显示，参与藏猪与约克生物学过程的多个基因都存在表达差异，包括先天免疫激活信号通道的受体和主要信号传递蛋白。