Huang Guan-Gen, Wang Hao-Yu, Wang Xiao-Han, Yang Tao, Zhang Xiao-Meng, Feng Chun-Lan, Zhao Wei-Min, Tang Wei
State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, PR China.
State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Phytomedicine. 2024 Mar;125:155343. doi: 10.1016/j.phymed.2024.155343. Epub 2024 Jan 8.
Zika virus (ZIKV) is a single-stranded RNA flavivirus transmitted by mosquitoes. Its infection is associated with neurological complications such as neonatal microcephaly and adult Guillain-Barré syndrome, posing a serious threat to the health of people worldwide. Therefore, there is an urgent need to develop effective anti-ZIKV drugs. Atranorin is a lichen secondary metabolite with a wide range of biological activities, including anti-inflammatory, antibacterial and antioxidant, etc. However, the antiviral activity of atranorin and underlying mechanism has not been fully elucidated.
We aimed to determine the anti-ZIKV activity of atranorin in human glioma cell line SNB-19 and investigate the potential mechanism from the perspective of viral life cycle and the host cell functions.
We first established ZIKV-infected human glioma cells (SNB-19) model and used Western Blot, RT-qPCR, immunofluorescence, fluorescence-activated cell sorting (FACS) and plaque assay to evaluate the anti-ZIKV activity of atranorin. Then we assessed the regulation effect of atranorin on ZIKV induced IFN signal pathway activation by RT-qPCR. Afterward, we introduced time-of-addition assay, viral adsorption assay, viral internalization assay and transferrin uptake assay to define which step of ZIKV lifecycle is influenced by atranorin. Finally, we performed virus infectivity assay, molecular docking and thermal shift assay to uncover the target protein of atranorin on ZIKV.
Our study showed that atranorin could protect SNB-19 cells from ZIKV infection, as evidenced by inhibited viral protein expression and progeny virus yield. Meanwhile, atranorin attenuated the activation of IFN signal pathway and downstream inflammatory response that induced by ZIKV infection. The results of time-of-addition assay indicated that atranorin acted primarily by disturbing the viral entry process. After ruling out the effect of atranorin on AXL receptor tyrosine kinase (AXL) dependent virus adsorption and clathrin-mediated endocytosis, we confirmed that atranorin directly targeted the viral envelope protein and lowered ZIKV infectivity by thermal shift assay and virus infectivity assay respectively.
We found atranorin inhibits ZIKV infection in SNB-19 cells via targeting ZIKV envelope protein. Our study provided an experimental basis for the further development of atranorin and a reference for antiviral drug discovery from natural resources.
寨卡病毒（ZIKV）是一种由蚊子传播的单链RNA黄病毒。其感染与新生儿小头畸形和成人吉兰-巴雷综合征等神经并发症相关，对全球人类健康构成严重威胁。因此，迫切需要开发有效的抗寨卡病毒药物。扁枝衣菌素是一种地衣次生代谢产物，具有广泛的生物活性，包括抗炎、抗菌和抗氧化等。然而，扁枝衣菌素的抗病毒活性及其潜在机制尚未完全阐明。
我们旨在确定扁枝衣菌素在人胶质瘤细胞系SNB-19中的抗寨卡病毒活性，并从病毒生命周期和宿主细胞功能的角度研究其潜在机制。
我们首先建立寨卡病毒感染的人胶质瘤细胞（SNB-19）模型，并使用蛋白质免疫印迹法、逆转录定量聚合酶链反应、免疫荧光、荧光激活细胞分选和蚀斑试验来评估扁枝衣菌素的抗寨卡病毒活性。然后，我们通过逆转录定量聚合酶链反应评估扁枝衣菌素对寨卡病毒诱导的干扰素信号通路激活的调节作用。随后，我们采用加样时间试验、病毒吸附试验、病毒内化试验和转铁蛋白摄取试验来确定寨卡病毒生命周期的哪个步骤受到扁枝衣菌素的影响。最后，我们进行病毒感染性试验、分子对接和热位移试验，以揭示扁枝衣菌素在寨卡病毒上的靶蛋白。
我们的研究表明，扁枝衣菌素可以保护SNB-19细胞免受寨卡病毒感染，病毒蛋白表达和子代病毒产量受到抑制证明了这一点。同时，扁枝衣菌素减弱了寨卡病毒感染诱导的干扰素信号通路激活和下游炎症反应。加样时间试验结果表明，扁枝衣菌素主要通过干扰病毒进入过程发挥作用。在排除扁枝衣菌素对AXL受体酪氨酸激酶（AXL）依赖性病毒吸附和网格蛋白介导的内吞作用的影响后，我们分别通过热位移试验和病毒感染性试验证实扁枝衣菌素直接靶向病毒包膜蛋白并降低寨卡病毒的感染性。
我们发现扁枝衣菌素通过靶向寨卡病毒包膜蛋白抑制SNB-19细胞中的寨卡病毒感染。我们的研究为扁枝衣菌素的进一步开发提供了实验依据，并为从自然资源中发现抗病毒药物提供了参考。
