Divergent mechanistic reliance on the phosphatidylinositol 4-kinase (PI4K)/oxysterol-binding protein (OSBP) axis during Zika virus versus dengue virus and West Nile virus replication organelle biogenesis.

Positive-strand RNA viruses remodel host endoplasmic reticulum (ER) membranes into replication organelles (ROs) that support genome replication. Members of the genus Orthoflavivirus exploit host lipid pathways, particularly the phosphatidylinositol 4-kinase (PI4K)-phosphatidylinositol 4-phosphate (PI4P)-oxysterol-binding protein (OSBP) axis, yet they display marked mechanistic divergence. Specifically, it raised a central and unresolved question: is local PI4P enrichment an indispensable determining factor for viral vesicle biogenesis, or is it merely a contributing factor to a more fundamental, underlying mechanical requirement for high membrane curvature? Zika virus (ZIKV) shows strong dependence on PI4P enrichment. ZIKV nonstructural protein 1 (NS1) binds negatively charged lipids, including PI4P, via a positively charged membrane-binding surface that includes Arg31. This interaction supports NS1-mediated ER remodeling and subsequent OSBP-mediated cholesterol delivery. In contrast, dengue virus (DENV) and West Nile virus (WNV) largely bypass strict PI4P dependence. Their RO biogenesis relies more heavily on viral membrane proteins (NS4A/NS4B), host reticulon RTN3.1A, alternative lipid-shape remodeling (including phospholipase A2-generated lysophosphatidylcholine in WNV, sterol availability, and fatty-acid synthesis. Beyond the PI4P-OSBP axis, virus-specific exploitation of lipid peroxidation (ALOX12 pathway), cholesteryl-ester-enriched lipid droplets (via SOAT1/SOAT2), nuclear lipid droplets, and glycerophospholipid/ceramide remodeling further differentiates these viruses. These distinctions have direct implications for host-targeted antivirals. PI4K and OSBP inhibitors show preferential activity against ZIKV, whereas SOAT1/2 inhibition exhibits broader anti-orthoflaviviral potential, and other targets (FASN, NAAA, ALOX12) display virus-weighted efficacy. Recognition of this mechanistic spectrum is essential for accurate interpretation of lipid-perturbation studies and for the rational design of broad-spectrum or virus-selective antiviral strategies.