Department of Infectious Diseases, Third Affiliated Hospital, 144991 Sun Yat-sen University, China.
Infectious Disease Center, 159355Guangzhou Eighth People's Hospital, Guangzhou Medical University, China.
Sci Prog. 2021 Oct;104(4):368504211058036. doi: 10.1177/00368504211058036.
This study is aimed at the determination of the effect of the immune-regulatory factor NLRX1 on the antiviral activity of hepatocytes against an external stimuli favoring hepatitis B virus infection, and to explore its mechanism of action.
A HepG2-NTCP model was established using the LV003 lentivirus. Cells were transfected using an overexpression vector and NLRX1 siRNA to achieve overexpression and interference of NLRX1 expression (OV-NLRX1, si-NLRX1). Levels of HBsAg and HBcAg were determined using Western blotting analysis and immunohistochemical analysis. The levels of hepatitis B virus DNA and hepatitis B virus cccDNA were determined by real-time quantitative polymerase chain reaction. The expression and transcriptional activity of IFN-α, IFN-β, and IL-6 were measured using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and promoter-luciferase reporter plasmids. Co-immunoprecipitation was used to determine the effect of NLRX1 on the interaction between MAVS and RIG-1. Western blotting was used to obtain the phosphorylation of essential proteins in the MAVS-RLRs signaling pathways.
NLRX1 promoted HepG2-NTCP cell hepatitis B virus infection. Compared to the control group, the levels of HBsAg, HBcAg, hepatitis B virus cccDNA, and hepatitis B virus DNA increased in the OV-NLRX1 group and decreased in the si-NLRX1. Co-immunoprecipitation results showed that NLRX1 competitively inhibited the interaction between MAVS and RIG-1, and inhibited the phosphorylation of p65, IRF3, and IRF7. Additionally, NLRX1 reduced the transcription activity and expression levels of the final products: IFN-α, IFN-β, and IL-6.
NLRX1 can counteract innate immune response induced by an external stimuli favoring hepatitis B virus infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells. Inhibition of the MAVS-RLR-mediated signaling pathways leads to a decline in the expression levels of I-IFN and IL-6.
本研究旨在确定免疫调节因子 NLRX1 对有利于乙型肝炎病毒感染的外部刺激物下肝细胞抗病毒活性的影响，并探讨其作用机制。
使用 LV003 慢病毒建立 HepG2-NTCP 模型。通过转染过表达载体和 NLRX1 siRNA 实现 NLRX1 表达的过表达和干扰（OV-NLRX1、si-NLRX1）。采用 Western blot 分析和免疫组织化学分析检测 HBsAg 和 HBcAg 的水平。采用实时定量聚合酶链反应检测乙型肝炎病毒 DNA 和乙型肝炎病毒 cccDNA 的水平。采用实时定量聚合酶链反应、酶联免疫吸附试验和启动子荧光素酶报告质粒检测 IFN-α、IFN-β 和 IL-6 的表达和转录活性。采用免疫共沉淀法确定 NLRX1 对 MAVS 和 RIG-1 相互作用的影响。采用 Western blot 法获得 MAVS-RLRs 信号通路中关键蛋白的磷酸化情况。
NLRX1 促进 HepG2-NTCP 细胞乙型肝炎病毒感染。与对照组相比，OV-NLRX1 组 HBsAg、HBcAg、乙型肝炎病毒 cccDNA 和乙型肝炎病毒 DNA 水平升高，si-NLRX1 组水平降低。免疫共沉淀结果表明，NLRX1 竞争性抑制 MAVS 和 RIG-1 的相互作用，抑制 p65、IRF3 和 IRF7 的磷酸化。此外，NLRX1 降低了最终产物 IFN-α、IFN-β 和 IL-6 的转录活性和表达水平。
NLRX1 可通过竞争抑制 HepG2-NTCP 细胞 MAVS-RLRs 信号通路，拮抗有利于乙型肝炎病毒感染的外部刺激诱导的固有免疫反应。抑制 MAVS-RLR 介导的信号通路导致 I-IFN 和 IL-6 的表达水平下降。
