<i>De novo</i> lipogenesis is required for HCV replication and release and supplies cellular lipid droplets that may contribute to viral assembly.

<p>(A) Effect on entry. Huh7.5.1 cells were treated with DMSO, 1 μM K1, or 100 nM soraphen A for 3 days after which the media was replaced with JFH-1 (MOI 0.1). HepG2 cells were used as a negative control. Cells were collected for qRT-PCR analysis at 1 hour post-infection. (B) Effect on replication. Huh7.5 cells harboring HCV subgenomic replicons were treated with DMSO, 1 μM K1, 100 nM soraphen A, or 250 nM of the NS5B polymerase inhibitor, sofosbuvir, for 3 days. HCV RNA was detected by qRT-PCR. (C) Effect on viral protein. Infected Huh7.5.1 cells were treated with DMSO, 1 μM K1, or 100 nM soraphen A for 3 days. Intracellular viral protein was assessed by immunoblotting. Densities of NS3 and core staining were calculated relative to vinculin and then normalized to DMSO. (D) Effect on translation. Huh7.5.1 cells were transfected with the bicistronic pFR_HCV_xb construct in which the HCV IRES regulated translation of renilla luciferase. Mock transfected cells served as controls. Twenty-four hours post-transfection, the media was replaced with DMSO, 1 μM K1, or 100 nM soraphen A for 3 days. Cellular lysates were assessed for both firefly and renilla luciferase activity. (E-G) Effect on assembly. Infected Huh7.5.1 cells were treated with DMSO, 1 μM K1 or 100 nM soraphen A for 3 days and stained for the nucleocapsid core protein and lipid droplets. Number of red (HCV core, F) and green (lipid droplets, G) was quantified over 10 fields in each experiment. Scale bar is equivalent to 20 μm. (H) Effect on infectious titer. Huh7.5.1 were infected with serially diluted supernatants of infected Huh7.5.1 cells that had been treated with DMSO, 1 μM K1, or 100 nM soraphen A for 3 days. The number of HCV-core positive focus forming units (FFU) was quantified 3 days post-infection. Results are the representative or mean ± SEM of 3–6 independent experiments. Statistical significance was calculated by one-way ANOVA with Tukey’s post-test. ns: not significant, <i>**p</i><0.01, ***<i>p</i><0.001.