宿主miR-10a的调控网络及其调控PRRSV感染与复制的分子机制

microRNA plays significant roles in virus-host interactions. Our previous project demonstrated that the expression level of miR-10a increased in porcine alveolar macrophages (PAMs) during porcine reproductive and respiratory syndrome virus (PRRSV) infection. Further miR-10a-5p inhibited PRRSV replication through suppression of signal recognition particle 14 (SRP14) expression. However, the transcriptional regulation of miR-10a itself, regulatory networks of miR-10 and its molecular mechanism in regulating PRRSV infection remain obscure. To determine the molecular mechanism through which PRRSV infection induced miR-10a expression, the transcription elements will be identified in the swine miR-10a gene promoter by way of bioinformatics analysis, luciferase reporter system, site-directed mutagenesis, electrophoretic mobility shift, and chromatin immunoprecipitation assays. Further, the miR-10a regulatory circuitry “transcription factor-miR-10a-SRP14” in porcine alveolar macrophages will be identified based on the miR-10a’s target SRP14 identified previously, and the molecular mechanism of miR-10a regulatory circuitry in regulating PRRSV infection and replication will also be elucidated. The proposed studies are expected to extend our understanding on the regulatory network of miR-10a and PRRSV infection mechanisms, and may also provide new molecular targets for developing preventive and control strategies against PRRSV.

microRNA在病毒-宿主互作网络中发挥极其重要调控作用。我们结题项目发现猪繁殖与呼吸综合征病毒（PRRSV）感染诱导猪肺泡巨噬细胞（PAM）的miR-10a上调表达，并初步证明其通过靶向信号识别颗粒SRP14抑制PRRSV复制。但miR-10a本身的表达调控机制、所参与的调控网络及其在PRRSV感染中的作用尚不清楚。本项目以miR-10a为核心，通过荧光素酶报告系统、定点突变、EMSA、ChIP等从上游鉴定调控miR-10a表达的转录因子，阐明miR-10a表达调控机制并回答PRRSV感染上调miR-10a原因；同时结合已从下游鉴定到的miR-10a靶基因SRP14，通过高表达、RNAi等解析PAM中的“转录因子-miR-10a-SRP14”调控回路并明确其在PRRSV感染中的作用。该研究将加深人们对miR-10a调控网络及PRRSV感染机制的认识，还可为PRRSV防控提供新靶点。

猪繁殖与呼吸综合征（PRRS）是对全球生猪产业危害极大的重大病毒性疫病。由于对PRRS病毒（PRRSV）感染与复制的分子调控机制缺乏深入认识，目前对该病的预防和控制尚未取得突破性进展。我们前期研究发现PRRSV感染诱导猪肺泡巨噬细胞（PAMs）中的miR-10a表达水平上调，并初步证明其通过靶向信号识别颗粒SRP14抑制PRRSV复制。但miR-10a本身的表达调控机制、所参与的调控网络及其在PRRSV感染过程中的作用尚不清楚。该项目克隆获得了猪miR-10a的启动子区，鉴定到了核心转录调控区，预测筛选了核心转录调控区的转录因子结合位点，发现转录因子IRF8能够特异性结合到猪miR-10a的启动子区抑制miR-10a的表达进而促进PRRSV复制；而PRRSV感染后，宿主细胞内IRF8的蛋白表达水平会显著降低，削弱了对miR-10a的负调控作用，导致miR-10a的表达水平升高；从microRNA转录后调控的角度发现miR-10a直接靶向SRP14的3’UTR，抑制SRP14的蛋白表达进而抑制PRRSV复制；发现了SRP14促进PRRSV复制，不是在病毒的吸附、入侵和释放阶段，而是PRRSV的非结构蛋白Nsp2通过蛋白互作将SRP14招募到PRRSV 转录起始复合体（RTCs）上，在病毒的复制阶段促进基因组的合成。研究成果对深入解析miR-10a调控PRRSV感染与复制的分子机制具有重要科学意义，也为确立miR-10a的调控通路作为PRRSV的防治靶点提供了科学依据。