Dengue virus suppression by 25-hydroxycholesterol is mediated by a combination of membrane fusion inhibition, innate immune activation and cholesterol biosynthesis dysregulation

Physicochemical properties and composition of cellular membranes are crucial for regulating broad cellular responses including signaling and defense against pathogens. Dengue virus (DENV) exploits cholesterol-rich membranes and host lipid pathways, such as cholesterol biosynthesis, lipid raft organization, and lipid droplet formation, for entry, replication, and assembly. Additionally, lipid-based plasma membrane signaling can trigger innate immune responses that attenuate viral growth, underscoring the dual role of lipids in facilitating and restricting DENV infection. Here, we demonstrate that 25-hydroxycholesterol (25-HC), an oxidized cholesterol metabolite, inhibits DENV infection through a multifaceted mechanism. 25-HC disrupts viral membrane fusion by altering cholesterol distribution and lipid raft organization, impairing the binding and fusion of the DENV envelope (E) protein with host membranes. Additionally, 25-HC modulates host cholesterol metabolism by suppressing biosynthesis pathways essential for viral replication while enhancing lipid droplet formation and stress-response pathways. Transcriptomic analyses reveal that 25-HC primes innate immune responses, activating proinflammatory pathways such as the NLRP3 inflammasome and MAPK signaling, while selectively modulating interferon-stimulated gene expression. Notably, 25-HC exhibits synergistic antiviral effects when combined with direct-acting antivirals like Remdesivir, underscoring its potential in combination therapies. These findings establish 25-HC as a promising candidate for host-directed antiviral strategies against DENV and other enveloped viruses.