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Tytuł:
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[Establishment of RIG-I knockout 293T cell line and its effect on the replication of influenza B virus].
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Autorzy:
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Tain L; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.; University of Chinese Academy of Sciences, Beijing 100049, China.
Jiao P; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.; College of Animal Science and Technology, Guangxi University, Nanning 530004, Guangxi, China.
Hou L; China Institute of Veterinary Drug Control, Beijing 100081, China.
Li Y; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Song Z; The High School Affiliated to Beijing Normal University, Beijing 100052, China.
Liu W; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.; University of Chinese Academy of Sciences, Beijing 100049, China.
Fan W; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.; University of Chinese Academy of Sciences, Beijing 100049, China.
Sun L; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.; University of Chinese Academy of Sciences, Beijing 100049, China.
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Źródło:
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Sheng wu gong cheng xue bao = Chinese journal of biotechnology [Sheng Wu Gong Cheng Xue Bao] 2020 Jan 25; Vol. 36 (1), pp. 109-121.
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Typ publikacji:
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Journal Article
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Język:
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Chinese
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Imprint Name(s):
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Original Publication: Beijing : Ke xue chu ban she,
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MeSH Terms:
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Influenza B virus*
DEAD Box Protein 58 ; HEK293 Cells ; Humans ; Immunity, Innate ; Interferons ; Virus Replication
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Contributed Indexing:
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Keywords: CRISPR/Cas9; RIG-I; gene knockout; influenza B virus
Local Abstract: [Publisher, Chinese] CRISPR/Cas9 基因编辑技术是通过人工设计的单向导RNA(Single-guide RNA，sgRNA)指导Cas9 蛋白对目的基因靶位点进行特异性的识别、结合和切割后，通过细胞的非同源末端连接或同源末端重组修复机制来完成对基因组的敲除与敲入的编辑技术。RIG-I 是机体的一种模式识别受体，能够识别胞质中的含5′-三磷酸基团的RNA，并通过与下游信号分子MAVS 相互作用，激活IRF3/7 和NF-κB，从而启动I 型干扰素和炎性因子的表达。已有研究表明，B 型流感病毒(IBV)在感染早期能够上调RIG-I 的表达水平。为了探索RIG-I 是否为B 型流感病毒激活抗病毒天然免疫信号通路的主要受体及其对IBV 复制的影响，本研究利用CRISPR-Cas9 技术对293T 细胞中的RIG-I 基因进行了敲除，经嘌呤霉素压力筛选到了一株稳定敲除RIG-I 基因的293T (RIG-I(-/-) 293T)细胞系。Western blotting 检测发现，IBV 或仙台病毒感染后该细胞系中RIG-I 不再表达，说明该敲除细胞系构建成功。IBV感染RIG-I(-/-) 293T 细胞后，干扰素、炎性因子及干扰素刺激基因的转录水平与野生型293T 细胞相比明显下降，并且在RIG-I(-/-) 293T 细胞中检测不到p65 和IRF3 磷酸化，表明IBV 感染早期细胞因子的表达主要依赖于RIG-I信号通路的激活。IBV 在野生型及RIG-I(-/-) 293T 细胞中的多步生长曲线表明，RIG-I 可抑制 IBV 的复制。以上结果表明，RIG-I 敲除的293T 细胞系构建成功，RIG-I 是IBV 激活下游抗病毒天然免疫信号通路的主要受体之一，且对IBV 的复制具有负调控作用，该研究为探索IBV 的感染机制奠定了基础。.
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Substance Nomenclature:
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9008-11-1 (Interferons)
EC 3.6.4.13 (DEAD Box Protein 58)
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Entry Date(s):
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Date Created: 20200220 Date Completed: 20200225 Latest Revision: 20200225
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Update Code:
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20240105
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DOI:
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10.13345/j.cjb.190176
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PMID:
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32072786
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The CRISPR/Cas9 gene editing technology directs Cas9 protein to recognize, bind and cleave the target site specifically by using artificial single-guide RNA (sgRNA), through non-homologous end joining or homologous end-recombinant repair mechanisms of cells, which can be engineered to knockout or knock-in of genomes. RIG-I is a pattern recognition receptor that recognizes the 5'-triphosphate-containing RNA in the cytoplasm and activates IRF3/7 and NF-κB by interacting with the downstream signaling molecule MAVS, thus initiating the expression of type I interferons and inflammatory factors. Previous studies found that influenza B virus (IBV) can up-regulate the expression of RIG-I. In the present study, to explore whether RIG-I is the major receptor for IBV to active the antiviral innate immune response and its effect on IBV replication, RIG-I gene in 293T cells was knocked out by CRISPR-Cas9 system, and a stable RIG-I knockout 293T (RIG-I(-/-) 293T) cell line was screened by puromycin pressure. The results of Western blotting showed that RIG-I was not expressed in this cell line after IBV or Sendai virus (SeV) infection, indicating that the RIG-I(-/-) 293T cell line was successfully constructed. The transcription levels of interferons, inflammatory factors and interferon-stimulated genes in RIG-I(-/-) 293T cells which were infected by IBV decreased significantly compared with those in wild-type 293T cells. Moreover, the phosphorylation of p65 and IRF3 were not detected in IBV or SeV infected RIG-I(-/-) 293T cells. It is indicated that the expression of cytokines mainly depends on the RIG-I-mediated signaling pathway at the early stage of IBV infection. Furthermore, the multi-step growth curves of IBV in the wild type and RIG-I(-/-) 293T cells showed that RIG-I inhibited the replication of IBV. Collectively, the RIG-I knockout 293T cell line was successfully constructed. We found that RIG-I is the main receptor for IBV to active the antiviral innate immune response and is critical for inhibiting IBV replication, which lays the foundation for further study of IBV infection mechanism.
