Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1

Abstract

Hepatitis C virus (HCV) infection is closely tied to the lipid metabolism of liver cells. Here we identify the triglyceride-synthesizing enzyme diacylglycerol acyltransferase-1 (DGAT1) as a key host factor for HCV infection. DGAT1 interacts with the viral nucleocapsid core and is required for the trafficking of core to lipid droplets. Inhibition of DGAT1 activity or RNAi-mediated knockdown of DGAT1 severely impairs infectious virion production, implicating DGAT1 as a new target for antiviral therapy.

Fig. 1. DGAT1 activity is necessary for HCV particle assembly at LDs

(a) Infection of shRNA-expressing Huh7.5 cells with low concentrations of eGFP-Jc1 viral stock. Spreading viral infection was measured by flow cytometry of eGFP (mean ± s.e.m.; n = 4; *p < 0.05, **p < 0.01). (b) Real-time RT-PCR analysis of DGAT1 or DGAT2 mRNAs in knockdown cells (mean ± s.e.m.; n = 4) or western blot analysis of DGAT1 protein expression. Available antibodies against DGAT2 do not reliably detect endogenous human DGAT2 in our hands. (c) Dose-dependent decrease of infectious titres in Huh7.5 cells transfected with Luciferase-Jc1 or Luciferase-JFH1 RNA and incubated with increasing concentrations of the DGAT1 inhibitor or DMSO for 48 h. Naïve Huh7.5 cells were infected with cell supernatants of treated cells and lysed 48 h post-infection to analyze luciferase activity (expressed as percent relative to DMSO control; mean ± s.e.m.: n = 3). (d) Dose-dependent decrease of infectious virus titers released from freshly isolated primary human hepatocytes infected with HCV-Jc1 viral stock and treated with increasing amounts of the DGAT1 inhibitor or DMSO for 3 days. Shown are infectivity titers (in FFU) expressed as percent of DMSO control. A single experiment is shown. (e) Real-time RT-PCR analysis of HCV RNA isolated from cells or from supernatants of Huh7.5 cells electroporated with eGFP-Jc1 RNA and treated with DGAT1 inhibitor (20 μM) or DMSO. Results are expressed as HCV RNA copy numbers per 1 μg total cellular RNA normalized to 18S rRNA (Intracellular) or per 1 ml culture supernatant (Secreted) at day 4 after transfection (mean ± s.d.; n = 6; **p < 0.01). (f) Western blot analysis of cell extracts described in e) lysed at day 4 after transfection. (g) Infection of naïve Huh7.5 cells with either intracellular or secreted viral particles isolated from Huh7.5 transfected with Luciferase-Jc1 RNA and treated with the DGAT1 inhibitor (20 μM) or DMSO. Shown are luciferase values expressed as percent of DMSO control (mean ± s.d.; n = 3; **p < 0.01). (h) Representative images and quantification of epifluorescence of Huh7 Lunet cells electroporated with eGFP-Jc1 RNA and treated with DMSO or DGAT1 inhibitor (20 μM) after oil-red-O (ORO) staining. LD area: mean of 1000 cells ± s.e.m.; LD diameter: mean of > 1600 LDs ± s.e.m.; LD number: mean of > 50 cells ± s.e.m. (i) Western blot analysis of cell extracts or isolated LD fractions from cells described in h). TG: extracted triglycerides analyzed by thin layer chromatography. (j) Indirect immunofluorescence of double-stranded RNA (α-dsRNA) at LDs (ORO) in cells described in h) (scale bar = 10 μm) and quantification of overlap of the signals for double-stranded RNAs and ORO per cell (mean of 30 cells ± s.e.m.).

Fig. 2. Specific interaction of the HCV core protein with DGAT1

(a) Coimmunoprecipitation assays in 293T cells transfected with expression vectors for the HCV core protein and Flag-DGAT1 or Flag-DGAT2 proteins and treated with DGAT1 inhibitor (20 μM) or DMSO. After immunoprecipitation with α-Flag agarose, the core protein was detected by western blotting with α-core antibodies. (b) Coimmunoprecipitation of the HCV core protein with endogenous DGAT1 in Huh7 hepatoma cells transduced with a core-expressing lentiviral vector. (c) Coimmunoprecipitation of the HCV core protein with endogenous DGAT1 in Huh7.5 and Huh7 Lunet cells electroporated with eGFP-Jc1 RNA. (d) Indirect immunofluorescence of core and endogenous DGAT1 in Huh7 cells transfected with wild-type or mutant (SPMT) core expression vectors (scale bar = 10 μm). (e) Coimmunoprecipitation assays in 293T cells transfected with expression vectors for wild-type (WT), mutant (SPMT) or truncated (1–173) core protein and the Flag-DGAT1-expressing plasmid. Arrows mark unprocessed (upper) and processed (lower) core protein. (f) Model of HCV core recruitment to DGAT1-generated LDs. See text for details.