Inhibition of enterovirus 71 replication and viral 3C protease by quercetin

BACKGROUND: Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date. METHODS: The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cᵖʳᵒ or 3Dᵖᵒˡ were tested. The interaction between EV71 3Cᵖʳᵒ and quercetin was predicted and calculated by molecular docking. RESULTS: Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cᵖʳᵒ, blocking viral replication, but not the activity of the protease, 2Aᵖʳᵒ, or the RNA polymerase, 3Dᵖᵒˡ. Modeling of the molecular binding of the 3Cᵖʳᵒ-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cᵖʳᵒ, blocking substrate recognition and thereby inhibiting EV71 3Cᵖʳᵒ activity. CONCLUSIONS: Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cᵖʳᵒ activity, thereby blocking EV71 replication.