上调 Kelch样 ECH相关蛋白 1 (KEAP1)抑制单纯疱疹病毒 1型复制

Upregulation of Kelch-like ECH-associated protein 1 represses the replication of herpes simplex virus type 1

摘要:

目的 探讨Kelch样ECH相关蛋白1 (Kelch-like ECH-associated protein 1, KEAP1)对单纯疱疹病毒1型(herpes simplex virus type 1, HSV-1)复制的影响，为抗单纯疱疹病毒研究提供理论依据。 方法 通过qPCR和Western blotting试验检测人视网膜色素上皮细胞(ARPE-19细胞)感染HSV-1后KEAP1-NRF2信号通路及病毒分子的mRNA和蛋白表达情况。构建KEAP1沉默和过表达的ARPE-19细胞株，采用Western blotting检测KEAP1沉默和过表达对核因子红细胞2相关因子2 (nuclear factor erythroid 2-related factor 2, NRF2)信号通路的影响。用KEAP1沉默和过表达细胞株感染HSV-1，采用qPCR检测病毒mRNA表达变化，采用免疫荧光和Western blotting检测病毒蛋白表达变化，采用空斑形成试验检测病毒滴度变化。用KEAP1沉默细胞株感染HSV-1，采用Western blotting检测不同时间点NRF2信号通路和病毒蛋白的表达情况。 结果 KEAP1沉默可激活NRF2信号通路，促进HSV-1的复制；KEAP1过表达可下调NRF2信号通路，抑制HSV-1的复制。这一结果与先前研究中关于NRF2信号通路上调及活化可抑制HSV-1复制的结论相矛盾。进一步研究发现，KEAP1沉默诱导的NRF2上调在HSV-1感染后受到显著抑制。 结论 KEAP1在宿主细胞抗HSV-1感染过程中发挥重要作用，其与NRF2的相互作用在抗病毒免疫应答中具有复杂的生物学功能。

Abstract:

Objective To investigate the effect of Kelch-like ECH-associated protein 1 (KEAP1) on the replication of herpes simplex virus type 1 (HSV-1) and thus provide theoretical support for anti-herpes simplex virus research. Methods The mRNA and protein levels of molecules in the KEAP1-NRF2 signaling pathway and viral molecules in ARPE-19 cells infected with HSV-1 were determined by qPCR and Western blotting, respectively. KEAP1-silenced and overexpressing ARPE-19 cell lines were constructed, and Western blotting was employed to assess the effects of KEAP1 silencing and overexpression on the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The KEAP1-silenced and overexpressing cell lines were subsequently infected with HSV-1. Changes in viral mRNA expression were detected via qPCR, while immunofluorescence and Western blotting were used to evaluate alterations in viral protein expression. Additionally, a plaque formation assay was conducted to measure variations in viral titer. Western blotting was performed on KEAP1-silenced cell lines infected with HSV-1 to assess the expression levels of NRF2 signaling pathway and viral proteins at different time points. Results Silencing of KEAP1 activated the NRF2 signaling pathway and promoted HSV-1 replication, whereas KEAP1 overexpression downregulated the NRF2 signaling pathway and inhibited HSV-1 replication. These findings contradict previous studies suggesting that upregulation and activation of the NRF2 signaling pathway can suppress HSV-1 replication. Further investigation revealed that KEAP1 silencing-induced NRF2 upregulation was significantly inhibited following HSV-1 infection. Conclusion KEAP1 plays a crucial role in the host cell resistance to HSV-1 infection, and its interaction with NRF2 exerts complex biological functions in antiviral immune responses.