Department of Biology, University of Rome Tor Vergata, Rome, Italy.
J Virol. 2013 Oct;87(20):11096-106. doi: 10.1128/JVI.01213-13. Epub 2013 Aug 7.
Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.
轮状病毒是一种无包膜病毒，具有独特的三层颗粒结构，内部包裹着分段的双链 RNA 基因组，其独特的形态发生途径需要形成细胞质包涵体，称为类质体，这一过程涉及非结构病毒蛋白 NSP5 和 NSP2。在这些结构中，11 条正链单链 RNA 协同包装和复制，生成病毒双链 RNA（dsRNA）基因组片段。轮状病毒感染是导致婴幼儿胃肠炎相关严重发病率和死亡率的主要原因，但目前尚无有效的抗病毒治疗方法。本文研究了噻唑烷类抗感染药物硝唑尼特的抗轮状病毒活性，并揭示了噻唑烷类药物抗轮状病毒的新机制。硝唑尼特及其活性代谢物替硝唑能抑制不同类型细胞中的猿猴 A/SA11-G3P[2]和人 Wa-G1P[8]轮状病毒复制，半数有效浓度（EC50）范围为 0.3 至 2μg/ml，半数细胞毒性浓度（CC50）大于 50μg/ml。噻唑烷类药物不影响病毒感染力、结合或进入靶细胞，也不会引起病毒蛋白表达的普遍抑制，而是会减小类质体的大小并改变其结构，减少轮状病毒 dsRNA 的形成。如蛋白/蛋白相互作用分析、共聚焦免疫荧光显微镜和类质体样结构形成分析所示，噻唑烷类药物通过阻碍非结构蛋白 NSP5 和 NSP2 之间的相互作用发挥作用。总之，这些结果表明，噻唑烷类药物通过干扰病毒形态发生抑制轮状病毒复制，可能代表一类针对轮状病毒胃肠炎的新型抗病毒药物。
