Mechanistic characterization of the antiviral effects of nordihydroguaiaretic acid against West Nile virus

Mosquito-borne arboviruses, such as West Nile virus (WNV), pose a global health threat and currently lack approved antiviral therapies. This unmet medical need underscores the importance of identifying novel therapeutic agents, with natural products emerging as promising candidates. Nordihydroguaiaretic acid (NDGA), a natural lignan from Larrea spp., is a known broad-spectrum antiviral. This study aimed to characterize the antiviral activity of NDGA against WNV and elucidate its mechanism of action, focusing on host cellular factors. Using plaque reduction and immunofluorescence assays in LLC-MK2 cells, we demonstrated that NDGA inhibits WNV replication with an EC₅₀ of 29.8 µM and a selectivity index of 3.9, showing no virucidal effect. Time-of-addition experiments revealed that NDGA exerts its inhibitory effect during the first two hours post-viral internalization, suggesting it targets early, intracellular events. We found that NDGA's antiviral activity is partly mediated by its inhibitory effect on the host's sterol regulatory element-binding protein (SREBP) pathway. Crucially, we provide novel evidence that NDGA treatment induces significant and quantifiable alterations in the host microtubule cytoskeleton, a network essential for Orthoflavivirus replication. These findings highlight NDGA as a promising host-targeting antiviral candidate for WNV and other related Orthoflaviviruses, acting through multiple mechanisms involving cytoskeletal reorganization.