miR-146a 通过 IRAK1/TRAF6/NF-κB 信号通路促进博尔纳病病毒 1 的复制。

miR-146a promotes Borna disease virus 1 replication through IRAK1/TRAF6/NF-κB signaling pathway.

机构信息

Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China; Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, China.

Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, China.

出版信息

Virus Res. 2019 Oct 2;271:197671. doi: 10.1016/j.virusres.2019.197671. Epub 2019 Jul 19.

DOI:10.1016/j.virusres.2019.197671

PMID:31330207

Abstract

BACKGROUND/AIMS: Borna disease virus 1 (BoDV-1) is a negative single-stranded RNA virus that is highly neurotropic. BoDV-1 infection can damage the central nervous system and cause inflammation. To survive in host cells, BoDV-1 must evade the host innate immune response. A previous study showed that miR-146a expression increased in neonatal rats infected with BoDV-1. miR-146a is a microRNA suggested to negatively regulate innate immune and inflammatory responses and antiviral pathways. Many groups have reported that its overexpression facilitates viral replication. However, it is unclear whether miR-146a is involved in escape from the host immune response during BoDV-1 infection.

METHODS

In this study, BoDV-1 was used to infect neonatal rats within 24 h of birth intracranially, as well as to infect human microglial cells (HMC3). miR-146a expression was analyzed by RT-qPCR. The TargetScanHuman database was used to find the target genes of miR-146a. A search of the binding sites of miR-146a and its target gene's 3'-untranslated region (3'UTR) was also performed using RNAhybrid software. The binding sites of miR-146a and the target gene's 3'UTR were detected by dual luciferase reporter assays. Overexpression and suppression studies of miR-146a were performed to determine its effect on BoDV-1 replication. The relative protein expression of members of the IRAK1/TRAF6/NF-κB signaling pathway was also evaluated by western blotting in HMC3.

RESULTS

After BoDV-1 infection of neurons in vivo and of HMC3 cells, miR-146a expression was significantly upregulated. miR-146a overexpression in HMC3 cells promoted viral replication, while its inhibition inhibited it. Through the TargetScanHuman database, we identified the target genes of anti-inflammatory miR-146a: IRAK1 and TRAF6. We also found that BoDV-1 could inhibit IRAK1 and TRAF6 expression in HMC3 cells. Moreover, we showed that the inhibition of IRAK1 and TRAF6 also led to decreases in the expression of P65 and phosphorylated P65 in the downstream NF-κB pathway. Subsequently, we confirmed the interaction of miR-146a with IRAK1 and TRAF6 by luciferase assay.

CONCLUSION

Our results suggest that miR-146a inhibits the IRAK1/TRAF6/NF-κB signaling pathway to facilitate BoDV-1 survival in host cells.

摘要

背景/目的：博尔纳病病毒 1（BoDV-1）是一种高度嗜神经的负单链 RNA 病毒。BoDV-1 感染可损害中枢神经系统并引起炎症。为了在宿主细胞中存活，BoDV-1 必须逃避宿主先天免疫反应。先前的研究表明，BoDV-1 感染的新生大鼠中 miR-146a 的表达增加。miR-146a 是一种被认为负调控先天免疫和炎症反应及抗病毒途径的 microRNA。许多研究小组报告说，其过表达促进了病毒的复制。然而，miR-146a 是否参与 BoDV-1 感染期间逃避宿主免疫反应尚不清楚。

方法

本研究采用新生大鼠在出生后 24 小时内经颅感染 BoDV-1 以及感染人小神经胶质细胞（HMC3）的方法，通过 RT-qPCR 分析 miR-146a 的表达。使用 TargetScanHuman 数据库寻找 miR-146a 的靶基因。使用 RNAhybrid 软件搜索 miR-146a 和其靶基因 3'UTR 的结合位点。通过双荧光素酶报告基因检测 miR-146a 和靶基因 3'UTR 结合位点。通过过表达和抑制 miR-146a 研究来确定其对 BoDV-1 复制的影响。还通过 Western blot 评估了 HMC3 中 IRAK1/TRAF6/NF-κB 信号通路成员的相对蛋白表达。

结果

BoDV-1 体内感染神经元和 HMC3 细胞后，miR-146a 的表达明显上调。HMC3 细胞中 miR-146a 的过表达促进了病毒的复制，而抑制其表达则抑制了病毒的复制。通过 TargetScanHuman 数据库，我们鉴定出抗炎 miR-146a 的靶基因：IRAK1 和 TRAF6。我们还发现 BoDV-1 可以抑制 HMC3 细胞中 IRAK1 和 TRAF6 的表达。此外，我们表明 IRAK1 和 TRAF6 的抑制也导致下游 NF-κB 通路中 P65 和磷酸化 P65 的表达减少。随后，我们通过荧光酶报告基因实验证实了 miR-146a 与 IRAK1 和 TRAF6 的相互作用。