Department of Biochemistry, German University in Cairo, New Cairo, Egypt.
Department of Microbiology and Immunology, German University in Cairo, New Cairo, Egypt.
J Gen Virol. 2021 Mar;102(3). doi: 10.1099/jgv.0.001571. Epub 2021 Mar 12.
The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments and thus helping virus assembly and release. This activity is essential for virus infectivity, making viroporins an attractive target for drug development. The protein sequence and drug sensitivity of p7 vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Here, we investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a-b, 2a-b, 3a and 4a using patch-clamp electrophysiology and cell-based assays. We established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity and its sensitivity to inhibitors. The results from the cell viability assay were consistent with control measurements using established assays of haemadsorption and intracellular pH, and agreed with data from patch-clamp electrophysiology. Hexamethylene amiloride (HMA) was the most potent inhibitor of p7 activity, but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. The alkyl-chain iminosugars B-DNJ, N-DNJ and N-DGJ were tested and their activity was found to be genotype-specific. In the current study, we introduce cell viability assays as a rapid and cost-efficient technique to assess viroporin activity and identify channel inhibitors as potential novel antiviral drugs.
丙型肝炎病毒 (HCV) 的 p7 病毒孔蛋白在感染病毒的细胞中形成一种细胞内质子传导跨膜通道，使细胞内隔室的 pH 值分流，从而帮助病毒组装和释放。这种活性对于病毒感染力至关重要，使病毒孔蛋白成为药物开发的有吸引力的靶标。p7 的蛋白序列和药物敏感性在 HCV 的七种主要基因型之间有所不同，但基本的通道活性得以保留。在这里，我们使用膜片钳电生理学和基于细胞的测定法研究了几种抑制剂对对应于基因型 1a-b、2a-b、3a 和 4a 的重组 HCV p7 通道的影响。我们建立了一种基于 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 的细胞活力测定法，用于评估重组 p7 在 HEK293 细胞中的表达及其对抑制剂的敏感性。该细胞活力测定法的结果与使用已建立的血球吸附和细胞内 pH 测定法的对照测量结果一致，并且与膜片钳电生理学的数据一致。己六亚甲基胺 (HMA) 是最有效的 p7 活性抑制剂，但在较高浓度下具有细胞毒性。金刚烷胺对所有基因型的 p7 均有效，而金刚乙胺的活性则取决于基因型。我们测试了烷基链亚氨基糖 B-DNJ、N-DNJ 和 N-DGJ，并发现其活性具有基因型特异性。在当前的研究中，我们引入了细胞活力测定法作为一种快速且具有成本效益的技术，用于评估病毒孔蛋白的活性，并确定作为潜在新型抗病毒药物的通道抑制剂。
