RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication.
RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication.
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DOI：
10.1186/s12985-018-1080-x
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发表时间：
2018-10-23
期刊：
影响因子：
4.8
通讯作者：
Zheng H
中科院分区：
文献类型：
--
作者：
Li P;Zhang X;Cao W;Yang F;Du X;Shi Z;Zhang M;Liu X;Zhu Z;Zheng H
维甲酸诱导基因I(RIG-I)是天然免疫系统重要的胞浆受体。塞尼卡谷病毒是一种新出现的感染猪的核糖核酸病毒，给养猪业造成了巨大的经济损失。RIG-I在不同的病毒感染过程中发挥不同的作用。RIG-I在SVV感染细胞中的作用尚不清楚。了解RIG-I在SVV感染中的作用将有助于阐明SVV在感染细胞中的感染过程。在本研究中，我们使用聚集的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白-9核酸酶(Cas9)基因组编辑工具建立了RIG-I基因敲除(KO)猪肾脏PK-15细胞系。采用Sanger测序法测定RIG-I基因序列，Western blotting检测RIG-I蛋白在RIG-I KO细胞中的表达。通过检测干扰素-β和干扰素刺激基因(ISGs)的基因表达水平，研究I型干扰素途径在仙台病毒或SVV感染的RIG-I KO细胞中的激活状态。用定量聚合酶链式反应、Western blotting、TCID50法和间接免疫荧光法检测病毒在RIG-I KO细胞中的复制状态。RIG-I在PK-15细胞中的基因编辑成功地破坏了RIG-I的表达。与野生型(WT)PK-15细胞相比，RIG-1KO PK-15细胞在模型病毒SEV刺激下，其干扰素-β和ISGs的表达水平较低。对感染病毒的RIG-I WT和KO细胞中病毒RNA和病毒蛋白的含量以及病毒产量进行了测定和比较，结果表明RIG-I基因敲除显著增加了SVV的复制和繁殖。同时，在病毒感染过程中，RIG-1KO细胞中干扰素-β和ISGs的表达明显低于RIG-I WT细胞。综上所述，本研究表明RIG-I对SVV具有抗病毒作用，并且在SVV感染过程中激活I型干扰素信号是必不可少的。此外，本研究还表明，CRISPR/Cas9系统可作为在SVV疫苗研制过程中修饰细胞系以提高病毒产量的有效工具。
Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells. In this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-β and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay. Gene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-β and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-β and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection. Altogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development.
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影响因子：
8.8
作者：
Feng Q;Hato SV;Langereis MA;Zoll J;Virgen-Slane R;Peisley A;Hur S;Semler BL;van Rij RP;van Kuppeveld FJ
通讯作者：
van Kuppeveld FJ
影响因子：
10.5
作者：
Iwasaki A
通讯作者：
Iwasaki A
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
4.6
作者：
Jorgensen, S. E.;Christiansen, M.;Mogensen, T. H.
通讯作者：
Mogensen, T. H.
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM