ZIKV NS3 disrupts Nrf2-mediated antioxidant defense and drives vascular smooth muscle cell damage and vascular dysfunction

Zika virus (ZIKV) infection has been linked to vascular complications, yet the basis of vascular injury after apparent clearance of circulating virus remains poorly understood. This study examined the temporal progression of ZIKV-associated vascular damage and defined the contribution of non-structural protein 3 (NS3) to oxidative stress-mediated vascular dysfunction. ZIKV infection was modeled in interferon-α/β receptor-deficient A129 mice and immunocompetent northern pig-tailed macaques (Macaca leonina). Vascular pathology and function were evaluated using histopathology, transmission electron microscopy, immunofluorescence, and vascular reactivity assays, and primary murine vascular smooth muscle cells were used for mechanistic analyses. Transcriptomic profiling, antioxidant pathway assessment, and co-immunoprecipitation assays were performed to identify NS3-dependent molecular events. In A129 mice, ZIKV remained detectable in blood and induced progressive vascular injury. In contrast, in immunocompetent M. leonina, circulating viral RNA declined to undetectable levels by 15 days post-infection (dpi); however, viral RNA and envelope protein remained detectable in vascular tissues at 63 dpi, indicating sustained vascular viral retention after blood clearance. Across both models, ZIKV infection caused sustained endothelial injury, increased migration of vascular smooth muscle cells, and impaired vascular contractile responses. Mechanistically, NS3 interacted with nuclear factor erythroid 2-related factor 2 (Nrf2), restricted Nrf2 nuclear translocation, and disrupted the Nrf2/Kelch-like ECH-associated protein 1/glutathione peroxidase 4 (Nrf2/KEAP1/GPX4) antioxidant signaling pathway, thereby increasing oxidative stress. Restoration of Nrf2 activity significantly improved antioxidant capacity and attenuated vascular injury. These findings indicate that vascular viral persistence may contribute to post-viremic vascular dysfunction and identify NS3-mediated suppression of Nrf2 signaling as a mechanism underlying ZIKV-induced vascular injury.