剪接因子3B在干扰素-α抗丙型肝炎病毒中的作用及机制研究

The precise mechanisms of Interferon alpha (IFN alpha) inhibits hepatitis C virus (HCV) have not been elucidated. In our previsous whole-genome siRNA high throughput screening, we have identified 93 genes that mediate IFN's anti-HCV effects. Many of the identified genes are involved in mRNA processing. For instance, one of the selected genes, splicing factor 3, subunit B (SF3B) is thought to be involved in mRNA processing, and its silencing abrogates IFN's suppressive effects against HCV. Splicing isoforms of IFN antivirual effector genes (for example, p46 and p52 OAS, etc.) have been demonstrated to exert different anti-HCV efficacy result from mRNA splicing variant products. Alternative splicing was the key player in producing the isoforms. The gene variant of HCV RNA and mutation rate had been associated with clinical reponse. There have been reports that alternative splicing modulating viral protein expression, mature RNA producding, and infectivity. Further characterization of SF3B on IFN's antiviral may reveal novel anti-HCV strategies. We now propose to build on these initial findings with the following Specific Aims. Aim 1. To identify the relationship between SF3B, alternative splicing, and HCV gene variants and splicing isoforms of IFN antivirual effector genes. We will test the effect of SF3B on IFN's antiviral in the JFH1 HCV infection model by performing siRNA SF3B knock down, overexpression SF3B, and application of SF3B inhibitor. The second-generation sequencing, exon microarray, quantitative PCR, Western blot, flow cytometry, immunofluorescence, gel shift assay, in vitro splicing, and immunoprecipitation will be employed to monitor the effects that SF3B regulates HCV replication through alternative splicing of IFN anti-viral effector genes. Aim 2. To determine the predicting value of SF3B in IFN alpha anti-HCV by investagting patient's serum, PBMC, and liver biospy samples. Our study would reveal the important role of SF3B in IFN alpha anti-HCV, suggest a new avenue for the mechanism research in IFN alpha anti-HCV, and provide a solid support in improving the therapy outcome of IFN alpha anti-HCV.

研究干扰素-α（Interferon α，IFN-α）抗丙型肝炎病毒（hepatitis C virus，HCV）无应答的机制有助于提高临床疗效。RNA干扰联合细胞平台的高通量筛选研究发现：信使RNA处理是与IFN-α抗HCV作用关系最密切的生物学过程、剪接因子3B（splicing factor 3B ，SF3B）通过与该生物学过程密切相关。本研究以JFH1（基因2a型HCV）感染的Huh7.5.1细胞为研究平台，用多种方法干扰SF3B基因表达，用第二代测序、外显子表达芯片、定量PCR、免疫印迹、细胞免疫荧光等方法，证明SF3B通过可变剪接调控病毒核酸的正确合成、IFN-α增强其调控作用；用体外剪接、迁移率变动、免疫共沉淀、流式细胞检测等方法，阐明该调控作用的机制；依据对患者临床疗效的研究，阐明SF3B可作为IFN-α抗HCV的疗效预测指标之一；为IFN-α抗HCV的机制研究提供新思路。

研究干扰素-α（Interferon α，IFN-α）抗丙型肝炎病毒（hepatitis C virus，HCV）无应答的机制有助于提高临床疗效。RNA 干扰联合细胞平台的高通量筛选研究发现：信使RNA 处理是与IFN-α抗HCV 作用关系最密切的生物学过程、剪接因子3B（splicing factor 3B ，SF3B）与该生物学过程密切相关。本研究以JFH1（基因2a 型HCV）感染的Huh7.5.1 细胞为研究平台，用特异性siRNA干扰SF3B基因的表达，用定量PCR、免疫印迹等方法，证明在IFN抗HCV作用中，SF3B1与IFN和HCV的相关度优于SF3B4；SF3B1有助于HCV Core蛋白（HCV的结构蛋白）的表达、但对HCV NS3蛋白（HCV的非结构蛋白）的影响不明显。沉默SF3B1后，干扰素效应蛋白（PKR、Mx1、OAS）的表达明显被抑制；NF-kB蛋白也明显被抑制；干扰素信号通路上的STAT1蛋白无明显变化、PI3K和MAPK蛋白表达增加。因此，SF3B1可能通过NF-kB促进HCV Core蛋白的合成。确切的机制研究是下一步工作的重点。本研究结果展示了SF3B1基因在HCV复制中的作用，为控制HCV复制提供了新思路。