MicroRNA-373通过负向调控Ⅰ型干扰素促进猪繁殖与呼吸综合征病毒（PRRSV）复制的机制研究

发布时间：2018-07-27 11:32

【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起母猪繁殖障碍和仔猪呼吸困难的高度传染性疾病。目前PRRS呈全球流行,这不仅给养猪业造成巨大的经济损失,而且也严重危害养猪业的健康发展。PRRSV之所以能够成功感染宿主并形成持续性感染,是因为PRRSV能够抑制或逃逸宿主先天性免疫和获得性免疫系统,尤其是PRRSV能够抑制I型干扰素(Type Ⅰ IFNs,IFN-I)的产生。因此,深入研究PRRSV免疫抑制和持续性感染的机制,将会为PRRS防控提供理论依据。MicroRNAs(miRNAs)是一类转录后调控基因表达的非编码小RNA,大量研究表明,miRNA通过靶向病毒基因组或调控宿主抗病毒反应,从而影响病毒的复制。已有研究表明,宿主miRNA表达谱的失衡可能与PRRSV引起的免疫抑制和持续性感染有关,然而,目前对PRRSV如何调控宿主miRNA表达的机制和宿主miRNA帮助病毒复制的机制尚不清楚。因此,本研究首先利用RNA高通量测序技术(RNA-Seq)发现PRRSV感染显著改变了MARC-145细胞miRNA表达谱,其中,以miR-373的表达量变化较为明显,进一步的荧光定量PCR(q RT-PCR)方法确证了PRRSV感染明显上调了MARC-145细胞中miR-373的表达量,那么PRRSV是如何上调miR-373的表达以及miR-373对PRRSV复制有何影响及其机理又是什么?本论文针对以上科学问题进行了详尽的研究,具体结果如下:PRRSV通过上调Sp1的表达而促进miR-373的转录。miR-373启动子截断、启动子序列的生物信息学分析和突变实验表明,Sp1(specificity protein 1,Sp1)是miR-373基因的重要转录因子,而EMSA和Ch Ip确证了Sp1能够直接与miR-373启动子结合。进一步实验发现PRRSV感染MARC-145细胞后,Sp1被明显上调,而Sp1 si RNA能明显抑制PRRSV对miR-373的上调表达,相反,过表达Sp1有助于PRRSV上调miR-373的表达,这表明PRRSV可以通过上调Sp1的表达而促进miR-373的转录。通过过表达病毒蛋白和启动子报告质粒等实验,发现PRRSV的非结构蛋白nsp9和结构蛋白N能够明显上调miR-373启动子活性和表达,而且nsp9和N蛋白也能促进Sp1的表达,这表明PRRSV通过nsp9和N蛋白上调Sp1的表达,进而促进miR-373转录。miR-373通过负向调控IFN-I信号通路而促进PRRSV复制。miR-373模拟物和抑制剂转染实验表明,转染miR-373模拟物的MARC-145细胞和PAMs其PRRSV载量明显增加,而miR-373抑制剂却明显抑制了PRRSV复制,这表明miR-373能够促进PRRSV复制。进一步研究发现Sp1能够明显促进PRRSV复制,而且这种促进作用是依赖于miR-373的表达。miR-373靶基因的筛选和鉴定结果表明,NFIA、NFIB、IRAK1、IRAK4、IRF1和IRF9是miR-373靶基因,由于IRAK1,IRAK4,IRF1是参与IFN-I产生的必需信号蛋白,且进一步研究结果表明miR-373可以抑制IFN-β的产生。因此,miR-373可以通过靶向IFN-I信号转导通路中关键分子NFIA、NFIB、IRAK1、IRAK4、IRF1和IRF9而抑制IFN-I信号转导通路,从而促进PRRSV复制。NFIA和NFIB促进IFN-I的产生和抑制PRRSV的复制。过表达NFIA和NFIB蛋白能够增强IFN-β启动子活性和表达水平,而下调NFIA和NFIB后可以抑制IFN-β启动子活性和表达水平,这表明NFIA和NFIB可能调控着IFN-β启动子活性和表达。随后的EMSA和Ch IP结果表明,NFIA/NFIB能与IFN-β启动子结合,以上结果表明NFIA和NFIB可能是IFN-β的转录因子进而能够调控IFN-β的转录表达。此外,过表达NFIA和NFIB后能够抑制PRRSV的复制,而下调NFIA和NFIB则可以促进PRRSV的复制,这表明NFIA和NFIB是可以抑制PRRSV复制的抗病毒蛋白。本研究首次发现NFIA和NFIB能够抑制RNA病毒(PRRSV)的复制。综上所述,本论文阐述了PRRSV调控并利用miR-373逃逸IFN-I抗病毒免疫反应,以及首次发现NFIA和NFIB能够调控IFN-I的产生和拮抗PRRSV的复制。本研究的结果对阐明PRRSV引起免疫抑制和持续性感染机制有着重要的参考意义,也为PRRS的防治研究提供新靶点。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) caused by the reproductive and respiratory syndrome virus (PRRSV) and the dyspnea of piglets. At present, PRRS is a global epidemic, which not only causes huge economic losses to the pig industry, but also seriously endangering pigs. The healthy development of.PRRSV is capable of successfully infecting the host and forming persistent infection, because PRRSV can inhibit or escape from the host's innate and acquired immune systems, especially the PRRSV that inhibits the production of Type I IFNs, IFN-I, and therefore studies the mechanism of PRRSV immunosuppression and persistent infection. It will provide a theoretical basis for PRRS prevention and control..MicroRNAs (miRNAs) is a non coding small RNA of a class of post transcriptional gene expression. A large number of studies have shown that miRNA affects virus replication by targeting the virus genome or regulating the host virus response, which has shown that the imbalance of the host miRNA expression spectrum may be immune to the immunity caused by PRRSV. Inhibition is associated with persistent infection, however, it is not clear how PRRSV regulates the host miRNA expression and the mechanism of host miRNA to help virus replication. Therefore, this study first uses RNA high throughput sequencing technology (RNA-Seq) to find that PRRSV infection significantly changes the miRNA expression profile of MARC-145 cells, in which the expression of miR-373 is measured. The further fluorescence quantitative PCR (Q RT-PCR) method confirms that PRRSV infection obviously up-regulated the expression of miR-373 in MARC-145 cells. Then, how does PRRSV increase the expression of miR-373 and what is the effect of miR-373 on PRRSV replication and what is its mechanism? This article has carried out a detailed study of the above scientific problems. The results are as follows: PRRSV promotes miR-373 transcriptional.MiR-373 promoter truncation by up regulation of the expression of Sp1. Bioinformatics analysis and mutation experiments of promoter sequences show that Sp1 (specificity protein 1, Sp1) is an important transcription factor of the miR-373 gene, and EMSA and Ch Ip confirm that it can be directly associated with the promoter. It was found that after PRRSV infection of MARC-145 cells, Sp1 was obviously up-regulated, while Sp1 Si RNA could obviously inhibit the up-regulated expression of PRRSV to miR-373. On the contrary, the overexpression of Sp1 contributed to the up regulation of miR-373 expression by PRRSV, which indicates that PRRSV can promote the transcription by up regulation of the expression. It is found that the non structural protein Nsp9 and structural protein N of PRRSV can obviously increase the activity and expression of miR-373 promoter, and Nsp9 and N protein can also promote the expression of Sp1, which indicates that PRRSV increases Sp1 expression through Nsp9 and N proteins and promotes the replication of miR-373 transcriptional transcription through negative regulation of the signaling pathway. 3 simulants and inhibitor transfection experiments showed that the PRRSV load of MARC-145 cells and PAMs transfected with miR-373 mimics increased significantly, while miR-373 inhibitors significantly inhibited PRRSV replication, which indicated that miR-373 could promote PRRSV replication. Further studies found that Sp1 could significantly promote PRRSV replication, and this promotion is dependent on miR-37. The results of the screening and identification of the target gene for the expression of.MiR-373 show that NFIA, NFIB, IRAK1, IRAK4, IRF1 and IRF9 are the target genes of miR-373, and as IRAK1, IRAK4, and IRF1 are the essential signaling proteins involved in the production of IFN-I, and further research shows that it can inhibit the production of the beta. Key molecules NFIA, NFIB, IRAK1, IRAK4, IRF1 and IRF9 inhibit the IFN-I signal transduction pathway, thus promoting PRRSV replication.NFIA and NFIB to promote IFN-I production and inhibition of PRRSV replication. Level, which indicates that NFIA and NFIB may regulate the activity and expression of IFN- beta promoter. Subsequent EMSA and Ch IP results indicate that NFIA/NFIB can be combined with IFN- beta promoter. The above results suggest that NFIA and NFIB may be the transcription factors of IFN- beta which can regulate the transcriptional expression of IFN- beta. The downregulation of NFIA and NFIB can promote the replication of PRRSV, which indicates that NFIA and NFIB are antiviral proteins that can inhibit PRRSV replication. This study first found that NFIA and NFIB can inhibit the replication of RNA virus (PRRSV). And NFIB can regulate the production of IFN-I and antagonize the replication of PRRSV. The results of this study have important reference significance for elucidating the mechanism of PRRSV induced immunosuppression and persistent infection, and also provide new targets for the prevention and control of PRRS.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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