Establishment of RIG-I knockout 293T cell line and its effect on the replication of influenza B virus

TIAN Lu1,2 JIAO Pengtao1,3 HOU Lidan4 LI Yun1 SONG Zhengyu5 LIU Wenjun1,2 FAN Wenhui1,2 SUN Lei1,2

(1.Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 100101)
(2.University of Chinese Academy of Sciences, Beijing, China 100049)
(3.College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China 530004)
(4.China Institute of Veterinary Drug Control, Beijing, China 100081)
(5.The High School Affiliated to Beijing Normal University, Beijing, China 100052)

【Abstract】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.

【Keywords】 CRISPR/Cas9; RIG-I; gene knockout; influenza B virus;

【DOI】

【Funds】 National Natural Science Foundation of China (No. 31672531) National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2018ZX10101004)

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(Translated by LU R)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 36, No. 01, Pages 109-121

January 2020

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Article Outline

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References