Identification of inhibitors of dengue viral replication using replicon cells expressing secretory luciferase
Identification of inhibitors of dengue viral replication using replicon cells expressing secretory luciferase
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DOI：
10.1016/j.antiviral.2019.104643
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发表时间：
2019-12-01
影响因子：
7.6
通讯作者：
Hishiki, Takayuki
中科院分区：
文献类型：
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作者：
Kato, Fumihiro;Nio, Yasunori;Hishiki, Takayuki
登革病毒(DENV)是登革热(DF)、登革出血热(DHF)和登革休克综合征(DSS)的病原体，在热带和亚热带地区仍然是一个公共卫生问题。然而，目前还没有针对DENV感染的抗病毒治疗。在这项研究中，我们的目标是建立一个稳定的报告复制子细胞系统，支持培养细胞中持续的病毒RNA复制。分离的复制子细胞在培养上清液中显示高水平的荧光素酶活性，同时在细胞中表达病毒编码的NS1、NS3和NS5蛋白。免疫荧光检测复制子细胞中的NS1、NS3蛋白和dsRNA。此外，抗DENV抑制剂利巴韦林和溴隐亭显著降低荧光素酶活性，且呈剂量依赖关系。用稳定转染复制子细胞的复合库进行高通量筛选，Z‘factor值为0.57。我们的筛选产生了几个候选化合物，包括一种已经显示出抗DENV活性的化合物。综上所述，我们的结果表明，这个表达分泌型荧光素酶基因的DENV亚基因组复制子细胞系统可以用于高通量筛选抗DENV化合物和分析DENV RNA的复制机制。
Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromo-criptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z' factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.