Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov;16(11):1295-8.
doi: 10.1038/nm.2238. Epub 2010 Oct 10.

Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1

Eva Herker  1 , Charles Harris, Céline Hernandez, Arnaud Carpentier, Katrin Kaehlcke, Arielle R Rosenberg, Robert V Farese Jr, Melanie Ott
Affiliations

Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1

Eva Herker et al. Nat Med. 2010 Nov.

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.

PubMed Disclaimer

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Fig. 1
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
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.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Moradpour D, Penin F, Rice CM. Nat Rev Microbiol. 2007;5:453–463. - PubMed
    1. Miyanari Y, et al. Nat Cell Biol. 2007;9:1089–1097. - PubMed
    1. Roingeard P, Hourioux C, Blanchard E, Prensier G. Histochem Cell Biol. 2008;130:561–566. - PubMed
    1. Boulant S, Targett-Adams P, McLauchlan J. J Gen Virol. 2007;88:2204–2213. - PubMed
    1. Huang H, et al. Proc Natl Acad Sci USA. 2007;104:5848–5853. - PMC - PubMed

Publication types

Substances