siRNA screen of the UFA biosynthesis pathway indicates that the rate-limiting enzyme is key for DENV2 replication.

(A) Huh7 cells were transfected with pools of four siRNAs targeting each gene and infected with DENV2. Infectious virus release was measured by plaque assay. (B) Validation of results using a single siRNA targeting SCD1 as well as indicated controls. (C) siRNAs were electroporated into Huh7 cells along with a luciferase-expressing DENV2 replicon. Viral RNA replication was measured by luciferase expression. (D) Huh7 cells were infected with DENV2 (MOI = 10) and cells were harvested at indicated time points and processed for gene expression, protein levels or enzymatic activity. Fold change in SCD1 mRNA in virus-infected cells was compared to mock-infected cells. qRT-PCR results are normalized to GAPDH. The same cells were flash frozen to preserve active enzymes. Cytoplasmic extracts were prepared and run on a western blot and probed for SCD1. Protein levels were normalized to actin. For enzyme activity measurements, TLC was carried out to measure conversion of 14C-labeled Stearoyl-CoA to 14C-labeled-oleic acid. The quantification of these 3 assays (mRNA, protein and SCD1 activity) is shown here. Results from a single representative experiment are presented. Three independent biological replicates showed similar temporal trends. (E) Huh7 cells were infected with DENV2, a UV-inactivated DENV2 or mock-infected. Five biological replicates were included at each time point. Metabolites were extracted and untargeted LC-MS was performed to measure the abundance of oleic acid at each time point. SCD1: stearoyl CoA desaturase, IRR: irrelevant siRNA (without a biological target for the siRNA sequence), DENV2: siRNA against dengue virus, serotype 2 genome. (A-C: one-way ANOVA with multiple comparisons tests: ** = p<0.05, *** = p<0.001, **** = p<0.0005, For E: DENV2 vs. mock: ** = p<0.001, **** = p<0.0001 and DENV2 vs UVI: ## = p<0.001, #### = p<0.0001).