Fig. 1: Neutral lipid–synthesizing enzymes are host dependency factors for ZIKV infection.

a Neutral lipid synthesis pathway: the diacylglycerol O-acyltransferases (DGAT) 1 and 2 use diacylglycerol and acyl-CoA as substrate to catalyze triglyceride (TG) formation. The sterol-O-acyltransferases (SOAT) esterify cholesterol. Neutral lipids are stored in cellular LDs. b Validation of shRNAs targeting DGAT1, DGAT2, or SOAT1 in ZIKV-infected cells. Huh7 cells were transduced with lentiviral particles encoding shDGAT1, shDGAT2, shSOAT1, or a non-targeting shRNA (shNT). Cells were infected at 3 days post transduction (dpt) with ZIKV (MOI 0.2), and total RNA was isolated at 48 hpi / 5 dpt. mRNA expression levels of the respective target were determined by quantitative RT PCR (qRT-PCR) (n = 3–4, *p ≤ 0.05, ***p ≤ 0.001, one-sample t-test). Knockdown of DGAT1 and SOAT1 was additionally confirmed by immunoblotting using SOAT1- and DGAT1-specific antibodies. GAPDH served as loading control. # indicates SOAT1-specific bands (n = 3). c Huh7 cells were transduced with lentiviral particles expressing shRNAs. 3 dpt, cells were infected with DENV (MOI 0.05), TBEV (MOI 0.05), WNV (MOI 0.001), or ZIKV (MOI 0.1). Supernatants were harvested at 48 hpi and titers were determined using TCID₅₀ (mean ± SEM, n = 3–4, *p ≤ 0.05, no asterisk = not significant, Welch´s t-test). Note that 3 replicates of shNT were included in each individual experiment. TG triglyceride, CE cholesterol ester. d Huh7 knockdown cells were infected with DENV (MOI 0.1), TBEV (MOI 0.1), WNV (MOI 0.001), or ZIKV (MOI 0.1) at 3 or 4 dpt. Lysates were harvested at 48 hpi and viral envelope (Flavi E) protein level were analyzed by immunoblotting. GAPDH served as loading control (Shown is one representative experiment, n = 2).