Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

基孔肯雅病毒（Chikungunya virus, CHIKV）是一种甲病毒（alphavirus），近期引发多起疫情暴发，因此被列为暂无有效治疗方案的再出现病原体。本研究构建了携带病毒复制酶蛋白，以及嘌呤霉素乙酰转移酶、增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein, EGFP）和海肾荧光素酶（Renilla luciferase）标记基因的CHIKV复制子。将该复制子转染至幼仓鼠肾细胞（Baby Hamster Kidney cell, BHK），获得稳定细胞系。通过筛选获得稳定生长的细胞株，其在非结构蛋白2（non-structural protein 2, nsP2）中存在Pro718→Gly的氨基酸替换以及5个氨基酸的插入，从而实现了非细胞病变表型。通过Northern印迹杂交（Northern blotting）对该复制子细胞系进行表征，发现其病毒RNA合成水平有所降低。该CHIKV复制子细胞系经验证可用于96孔板格式的抗病毒筛选，并被用于针对356种化合物（天然产物及临床获批药物）的聚焦筛选。研究发现，5,7-二羟基黄酮类化合物芹菜素（apigenin）、白杨素（chrysin）、柚皮素（naringenin）与水飞蓟宾（silybin）可抑制CHIKV复制子表达的EGFP及海肾荧光素酶（Rluc）标记基因的活性。在针对赛姆利基森林病毒（Semliki Forest virus, SFV）的平行筛选中，上述化合物的抗甲病毒活性得到验证，同时还鉴定出数种对SFV的半最大效应浓度（half maximal inhibitory concentration, IC50）介于0.4~24 µM之间的新型抑制剂。基于温度敏感型SFV突变株的新型侵入实验显示，氯丙嗪（chlorpromazine）与另外5种带有10H-吩噻嗪基结构的化合物可抑制SFV的细胞侵入过程。此类化合物还可减轻SFV及辛德毕斯病毒（Sindbis virus）诱导的细胞病变效应（cytopathic effect, CPE），并在病毒收获实验中抑制SFV病毒粒子的产生。最后，研究采用具有感染性的CHIKV验证了所选化合物的抗病毒效果。综上，本研究开发的甲病毒抑制剂筛选策略，不仅鉴定出可用于开发甲病毒侵入与复制阶段抑制剂的潜在先导结构，同时证实了CHIKV复制子与SFV可作为抗CHIKV筛选的生物安全替代模型的实用性。