USP26负性调控RLR信号通路对肠道病毒71型复制的影响*

摘要:目的研究泛素特 异性蛋白酶26( ubiquitin-specific protease 26, USP26)通过调控RLR信号通路影响肠道病毒71型(EV71)感染的机制和功能，深入了解EV71感染及其逃逸免疫防御关系,为临床治疗EV71 感染提供依据。方法利用RT-PCR和Western blot 分别检测EV71感染横纹肌肉瘤细胞(RD)0、2.4、8、12、24 h后的USP26 mRNA、VPI mRNA及其蛋白质的表达水平;在RD细胞中设置转染USP26-siRNA( 实验组)和转染阴性对照siRNA(对照组),再用EV71感染RD细胞,收集感染8、12和24 h时的mRNA样本,RT-PCR检测VPI mRNA的表达情况;收集感染8 h时的蛋白质样本，Western blot 检测细胞中MDAS5、p-IRF3、IRF3蛋白的表达水平并计算p-IRF3/IRF3的相对比值;利用空斑试验检测病毒滴度水平。结果Westem blot 和RT-PCR结果表明,EV71感染过程中USP26的表达上调(P<0.05);RD细胞转染后实验组EV71中VPI mRNA的表达水平明显低于同一时间的对照组(P<0.05),感染8h实验组中MDA5蛋白和p-IRF3蛋白的表达水平均显著高于对照组(P<0.05);两组RF3总蛋白的表达水平差异无统计学意义;空斑试验中实验组EV71的病毒滴度显著低于对照组(P<0.05)。结论USP26 可通过负性调控RLR信号通路参与抗病毒免疫反应，敲减USP26可抑制EV71在细胞中的复制。

USP26 negatively regulates RLR signaling pathway and affects replication of Enterovirus type 71

Abstract: Objective To study the mechanism and function of deubiquitinase USP26 ( ubiquitin-specifie protease 26) affecting enterovirus type 71 ( EV71) infection by regulating RLR ( retinoid acid-inducible gene-I-like receptor) signaling pathway and the relationship between EV71 infection and its escaping immune defense in more depthin order to provide new theoretical evidences for the clinical treatment of EV71 infection. Methods RT-PCR was used to detect the expression levels of USP26 and VPI mRNA in EV71-infected rhabdomyosarcoma cells (RD) at0, 2, 4, 8, 12 and 24 h, and Western blot was used to detect the expression levels of corresponding proteins respectively. USP26-siRNA ( experimental group) and negative contol siRNA ( contol group ) were transfected into RD cells respectively, and then EV71 were subsequently infected into the RD cells. The samples were collected at8, 12 and 24 h, and the expression levels of VPI mRNA in the samples were detected by RT-PCR. The samples were collcted in 8 hours after EV71 infection, and the expression levels of MDA5, p-IRF3 and IRF3 proteins in the cells were detected by Western blot. The relative ratio of pIRF3/IRF3 was calculated. The viral titer levels were detected using spot experiments. Results Western blot and RT-PCR results showed that the expression of USP26 was upregulated in the RD cells during EV71 infection (P<0.05). After transfection of EV71 in RD cells, the expression of VP1 mRNA in the EV71 experimental group was significantly lower than that in the control group at the same time ( P<0.05), and the expression levels of MDA5 and p-IRF3 protein in the experimental group were higher than those in the control group at 8 h ( P<0.05 ). There was no statistically significant difference for the protein level of IRF3 between the two groups. In plaque experiment, the virus titer of EV71 in the experimental group was significantly lower than that in the control group ( P<0.05).Conclusion USP26 may participate in antiviral immune responses by negatively regulating RLR signaling pathway, and knocking down USP26 could inhibit EV71 replication in cells.