Pacheco Beatriz, Fernández-Oliva Alberto, García-Serradilla Moisés, Risco Cristina
Cell Structure Laboratory, National Center for Biotechnology, National Research Council, CNB-CSIC, Darwin 3, 28049 Madrid, Spain.
Department of Biochemistry and Molecular Biology, School of Pharmacy, Universidad Complutense de Madrid, Plaza Ramon y Cajal s/n, 28040 Madrid, Spain.
J Gen Virol. 2023 Apr;104(4). doi: 10.1099/jgv.0.001838.
Drug repurposing is a valuable source of new antivirals because many compounds used to treat a variety of pathologies can also inhibit viral infections. In this work, we have tested the antiviral capacity of four repurposed drugs to treat Bunyamwera virus (BUNV) infection in cell cultures. BUNV is the prototype of the order, a large group of RNA viruses that includes important pathogens for humans, animals and plants. Mock- and BUNV-infected Vero and HEK293T cells were treated with non-toxic concentrations of digoxin, cyclosporin A, sunitinib and chloroquine. The four drugs inhibited BUNV infection with varying potency in Vero cells, and all except sunitinib also in HEK293T cells, with digoxin rendering the lowest half maximal inhibitory concentration (IC). Since digoxin rendered the best results, we selected this drug for a more detailed study. Digoxin is an inhibitor of the Na/K ATPase, a plasma membrane enzyme responsible for the energy-dependent exchange of cytoplasmic Na for extracellular K in mammalian cells and involved in many signalling pathways. Digoxin was shown to act at an early time point after viral entry reducing the expression of the viral proteins Gc and N. Effects on the cell cycle caused by BUNV and digoxin were also analysed. In Vero cells, digoxin favoured the transition from G1 phase of the cell cycle to S phase, an effect that might contribute to the anti-BUNV effect of digoxin in this cell type. Transmission electron microscopy showed that digoxin impedes the assembly of the characteristic spherules that harbour the BUNV replication complexes and the morphogenesis of new viral particles. Both BUNV and digoxin induce similar changes in the morphology of mitochondria that become more electron-dense and have swollen cristae. The alterations of this essential organelle might be one of the factors responsible for digoxin-induced inhibition of viral infection. Digoxin did not inhibit BUNV infection in BHK-21 cells that have a digoxin-resistant Na/K ATPase, which suggests that the effects of the blockade of this enzyme is a key factor of the antiviral activity of digoxin in BUNV-infected Vero cells.
药物重新利用是新抗病毒药物的一个有价值来源，因为许多用于治疗各种病症的化合物也能抑制病毒感染。在这项工作中，我们测试了四种重新利用的药物在细胞培养物中治疗布尼亚姆韦拉病毒（BUNV）感染的抗病毒能力。BUNV是该目病毒的原型，该目是一大类RNA病毒，包括对人类、动物和植物都很重要的病原体。用无毒浓度的地高辛、环孢素A、舒尼替尼和氯喹处理未感染和感染BUNV的Vero细胞和HEK293T细胞。这四种药物在Vero细胞中以不同效力抑制BUNV感染，除舒尼替尼外，在HEK293T细胞中也有抑制作用，地高辛的半数最大抑制浓度（IC）最低。由于地高辛效果最佳，我们选择该药进行更详细的研究。地高辛是钠钾ATP酶的抑制剂，钠钾ATP酶是一种质膜酶，负责在哺乳动物细胞中进行能量依赖的细胞质钠与细胞外钾的交换，并参与许多信号通路。结果表明，地高辛在病毒进入后的早期时间点起作用，降低病毒蛋白Gc和N的表达。还分析了BUNV和地高辛对细胞周期的影响。在Vero细胞中，地高辛促进细胞周期从G1期向S期转变，这种作用可能有助于地高辛在此细胞类型中发挥抗BUNV的作用。透射电子显微镜显示，地高辛阻碍含有BUNV复制复合物的特征性小球的组装以及新病毒颗粒的形态发生。BUNV和地高辛都会使线粒体形态发生类似变化，线粒体变得电子密度更高且嵴肿胀。这种重要细胞器的改变可能是地高辛诱导病毒感染抑制的因素之一。地高辛在具有对地高辛耐药的钠钾ATP酶的BHK-21细胞中不抑制BUNV感染，这表明阻断该酶的作用是地高辛在感染BUNV的Vero细胞中抗病毒活性的关键因素。
