Banducci-Karp, A.; Brixton, S.; Shah, P. M.; Li, M.-Y.; Bancroft, J.; Riepsaame, J.; Dhaliwal, R.; Melia, C. E.; Malavige, G. N.; Sanyal, S.; Bancroft, J.

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the molecular mechanisms governing lipid metabolism during infection remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2) as critical host dependency factors for flavivirus infection. SOAT1/2 activities were upregulated early during infection, coinciding with increased LD formation, which underwent transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic ~100-fold reduction in viral production. Mechanistically, SOAT1/2 generate cholesteryl ester-enriched lipid droplets with fundamentally altered proteomes, enriched in fatty acid remodelling enzymes, Rab-GTPases, lipid transport proteins and sphingomyelinases. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. SOAT1/2 deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the clinical relevance of viral lipid exploitation, analysis of dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever, compared to lean patients. This study establishes SOAT1/2 as essential host factors that enable flavivirus morphogenesis through specialised cholesteryl ester-enriched LDs, revealing a previously unrecognised virus-host interaction mechanism and identifying host lipid metabolism as a promising therapeutic target for combating flavivirus infections.