Source data for Fig 4.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that has re-emerged to cause large outbreaks of human infections worldwide. There are currently no approved antivirals for treatment of CHIKV infection. Recently, we reported that the ribonucleoside analog 4′-fluorouridine (4′-FlU) is a highly potent inhibitor of CHIKV replication, and targets the viral nsP4 RNA dependent RNA polymerase. In mouse models, oral therapy with 4′-FlU diminished viral tissue burdens and virus-induced disease signs. To provide critical evidence for the potential of 4′-FlU as a CHIKV antiviral, here we selected for CHIKV variants with decreased 4′-FlU sensitivity, identifying two pairs of mutations in nsP2 and nsP4. The nsP4 mutations Q192L and C483Y were predominantly responsible for reduced sensitivity. These variants were still inhibited by higher concentrations of 4′-FlU, and the mutations did not change nsP4 fidelity or provide a virus fitness advantage in vitro or in vivo. Pathogenesis studies in mice showed that the nsP4-C483Y variant caused similar disease and viral tissue burden as WT CHIKV, while the nsP4-Q192L variant was strongly attenuated. Together these results support the potential of 4′-FlU to be an important antiviral against CHIKV.

基孔肯雅病毒（Chikungunya virus, CHIKV）是一类致关节炎甲病毒，近年来再度流行，在全球范围内引发多起大规模人类感染暴发疫情。目前尚无获批用于临床治疗CHIKV感染的抗病毒药物。本团队此前报道显示，核苷类似物4′-氟尿苷（4′-fluorouridine, 4′-FlU）可强效抑制CHIKV复制，其作用靶点为病毒的nsP4（非结构蛋白4）RNA依赖的RNA聚合酶。在小鼠感染模型中，口服给予4′-氟尿苷可显著降低病毒组织载量，并缓解病毒诱导的疾病症状。为进一步验证4′-FlU作为CHIKV抗病毒药物的应用潜力，本研究筛选获得对4′-FlU敏感性降低的CHIKV变异株，鉴定出nsP2（非结构蛋白2）与nsP4上的两对突变位点；其中nsP4上的Q192L与C483Y突变是导致病毒对4′-FlU敏感性下降的主要原因。该类变异株在更高浓度的4′-FlU处理下仍可被有效抑制，且上述突变未改变nsP4的复制保真度，亦未在体外细胞培养或体内小鼠模型中为病毒带来增殖适合度优势。小鼠致病机制研究显示，nsP4-C483Y变异株引发的疾病症状与病毒组织载量均与野生型（wild type, WT）CHIKV无显著差异，而nsP4-Q192L变异株则表现出明显的减毒特征。综上，本研究结果证实4′-FlU具备成为抗CHIKV重要抗病毒药物的潜力。