Myricetin prevents lethal enterovirus infection by broadly targeting a conserved surface-exposed loops on the VP1...

Enteroviruses impose a significant global health burden, causing outbreaks of Hand, Foot, and Mouth Disease (HFMD) and severe neurological complications. Currently, no specific and broadly effective antiviral therapeutics are available due to the rapid mutation and co-circulation of diverse viral strains. The viral capsid protein VP1 is critical for viral integrity and host cell entry, making it an attractive target for drug development. Myricetin (MC) is a highly safe, naturally occurring flavonol widely distributed in medicinal plants (e.g., Myrica rubra) with well-documented ethnopharmacological applications and antiviral properties. However, its specific interaction with non-enveloped enteroviruses remains elusive.

This study aimed to comprehensively investigate the broad-spectrum antiviral efficacy of MC against diverse, highly pathogenic enteroviruses, structurally and functionally elucidate its molecular mechanism targeting the VP1 capsid protein, and evaluate its preclinical therapeutic potential in vivo.

The antiviral activity of MC was evaluated in vitro using cytopathic effect (CPE) reduction assays, plaque viral reduction assays, and RT-qPCR against a diverse panel of viral strains (EV-A71, CV-A16, EV-D68, CV-B3, CV-A6, and human coronavirus OC43 [HCoV-OC43]). The mechanism of action was investigated utilizing time-of-addition and temperature-shift attachment assays. Target engagement was mapped through continuous drug-resistance selection, whole-genome sequencing, and reverse genetics (via a mutant reporter virus construction). Structural interactions were modeled via molecular docking, 100-ns molecular dynamics (MD) simulations, and the binding affinity was quantitatively validated using Biolayer Interferometry (BLI). In vivo efficacy was assessed in a lethal EV-A71-infected neonatal ICR mouse model.

MC exhibited robust, broad-spectrum antiviral activity against all tested enteroviruses (IC₅₀ in the low micromolar range) with a favorable, dose-dependent safety margin (high CC₅₀). Time-of-addition and temperature-shift assays revealed that MC functions as a viral entry inhibitor, directly blocking the initial attachment of the virus to host cells. Crucially, viral passaging identified a non-synonymous E98K escape mutation on the EV-A71 VP1 capsid protein, which was proven via reverse genetics to completely abrogate the antiviral efficacy of MC. BLI analysis confirmed a direct, strong interaction (Kd = 16.66 µM) between MC and purified VP1. Computational analyses elucidated this broad-spectrum capability, revealing that MC specifically binds to a conserved surface-exposed region formed by the flexible BC, DE, and HI loops within the VP1 protein, distinct from classical deep-pocket binders. In vivo, systemic administration of MC (100 mg/kg) successfully rescued mice from lethal EV-A71 infection, alleviating extreme weight loss and severe hind-limb paralysis, while significantly clearing viral RNA loads from the brain, lungs, and muscle tissues.

MC acts as a novel, naturally derived, broad-spectrum enterovirus capsid binder that physically intercepts viral entry by targeting evolutionarily conserved surface-exposed loops on the VP1 protein. Supported by clear ethnopharmacological relevance, definitive mechanistic clarity, and robust translational efficacy in vitro and in vivo, MC represents a highly valuable natural antiviral lead for the management of current and emerging enterovirus outbreaks.