RIG-I敲除293T细胞系的建立及其对B型流感病毒复制的影响

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

摘要:

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。

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.