Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins

Abstract

Hepatitis C virus (HCV) and triglyceride-rich very low-density lipoproteins (VLDLs) both are secreted uniquely by hepatocytes and circulate in blood in a complex. Here, we isolated from human hepatoma cells the membrane vesicles in which HCV replicates. These vesicles, which contain the HCV replication complex, are highly enriched in proteins required for VLDL assembly, including apolipoprotein B (apoB), apoE, and microsomal triglyceride transfer protein. In hepatoma cells that constitutively produce infectious HCV, HCV production is reduced by two agents that block VLDL assembly: an inhibitor of microsomal triglyceride transfer protein and siRNA directed against apoB. These results provide a possible explanation for the restriction of HCV production to the liver and suggest new cellular targets for treatment of HCV infection.

Fig. 1.

Localization of HCV NS and cellular proteins in vesicles purified from Huh7 cells harboring HCV replicons. On day 0, Huh7-5A-GFP-6 cells (in which NS5A contains a Flag epitope tag) and Huh7-K2040 cells (in which NS5A is not epitope-tagged) were set up at 7 × 10⁵ cells per 60-mm dish. On day 2, cells were harvested and homogenized. The cell homogenates were incubated with control antibody IgG-2001 or anti-Flag-coated Dynabeads as indicated and subjected to magnetic immuno-isolation as described in Materials and Methods. (A) Lysates of bound and unbound vesicles derived from the same amount of cells (0.05–0.2 per dish) were subjected to SDS/PAGE followed by immunoblot analysis with antibodies reacting against the indicated proteins. Filters were exposed for 10–30 s. (B) Bound and unbound vesicles were solubilized with buffer C, and protein concentration in the detergent lysate was determined by the BCA Protein Assay Kit (Pierce). Each value is the mean of triplicate measurements in the same experiment. Similar results were obtained in more than three independent experiments.

Fig. 2.

Localization of HCV and cellular RNA in vesicles purified from Huh7 cells harboring HCV replicons. On day 0, Huh7, Huh7-5A-GFP-6 cells (in which NS5A contains a Flag epitope tag), and Huh7-K2040 cells (in which NS5A is not epitope-tagged) were set up at 7 × 10⁵ cells per 60-mm dish. On day 2, cells were harvested and homogenized. The cell homogenates were incubated with control antibody IgG-2001 or anti-Flag-coated Dynabeads as indicated and then subjected to magnetic immuno-isolation. RNA was then extracted from bound vesicles derived from two dishes of cells and unbound fractions derived from 1/3 dish of cells and then subjected to RT-PCR analysis with primers specific to HCV RNA (A) and quantitative real-time PCR analysis with the indicated primers (B). Each value in B is the mean of triplicate measurements in the same experiment. An asterisk denotes the level of statistical significance (Student's test) between values obtained from the experiment in which anti-Flag-coated Dynabeads were incubated with cell homogenates containing Flag-tagged NS5A and control experiments in which either anti-Flag-coated Dynabeads or the Flag epitope on NS5A was absent. ∗, P < 0.005. Similar results were obtained in one other independent experiment.

Fig. 3.

Localization of cellular proteins involved in VLDL assembly in vesicles purified from Huh7 cells harboring HCV replicons. Cells were set up, harvested, and analyzed the same way as described in Fig. 1A, except that the immunoblot analysis was performed with antibodies reacting against the indicated proteins. Filters were exposed for 5–30 s.

Fig. 4.

Colocalization of apoB with NS5A in Huh7 cells harboring HCV replicons. On day 0, Huh7.5 cells (A–C) and Huh7-5A-GFP-6 (D–F) cells were set up at 1 × 10⁵ cells per well in a six-well plate. On day 2, cells were fixed, stained with anti-GFP (A and D) or anti-apoB (B and E), and subjected to multicolored immunofluorescent microscopy as described in Materials and Methods. (C) A merged fluorescent composite images of A and B. (F) A merged fluorescent composite images of D and E. Within each merged composite image, yellow denotes protein colocalization. (Magnification: ×63.)

Fig. 5.

Decreased secretion of infectious HCV particles from Huh7-GL cells treated with MTP inhibitor. On day 0, Huh7-GL cells were set up at 7 × 10⁵ cells per 60-mm dish. On day 1, cells were treated with (+) or without (−) 100 nM of the MTP inhibitor, BMS-2101038. Sixteen hours later on day 2, cells were switched to serum-free medium in the absence (−) or presence (+) of the same amount of the MTP inhibitor. (A) After incubation for the indicated time, the culture medium was harvested and subjected to SDS/PAGE followed by immunoblot analysis with the indicated antibodies. Filters were exposed for 30 s. (B) After incubation for 4 h, cells and culture medium were harvested. HCV RNA copy numbers and titers in the medium were determined as described in Materials and Methods. Total RNA was prepared from cells and subjected to first-strand cDNA synthesis and quantitative real-time PCR analysis. Values (mean ± SD of three measurements in the same experiment) are plotted relative to the control that was not treated with the MTP inhibitor, which was set at 1. Asterisks denote the level of statistical significance (Student's test) between control cells and cells treated with the MTP inhibitor. ∗, P < 0.005; ∗∗, P < 0.05. HCV copy number and titer in control cells were 7 × 10⁵/ml and 3.5 × 10² foci formation units/ml, respectively. Similar results were obtained in more than five independent experiments.

Fig. 6.

Decreased secretion of infectious HCV particles from Huh7-GL cells treated with siRNA targeting apoB. On day 0, Huh7-GL cells were set up at 2 × 10⁵ cells per 60-mm dish. On day 1, cells were transfected with 400 pmol per dish of siRNA duplexes targeting GFP or apoB as indicated. On day 4, cells were switched to serum-free medium. (A) After incubation for 4 h, cells were harvested, after which total RNA was prepared and subjected to first-strand cDNA synthesis and quantitative real-time PCR analysis. Values shown are relative to the GFP siRNA-transfected control, which was set to 1. (B–D) After incubation for the indicated amount of time, culture medium was harvested. (B) The medium was subjected to SDS/PAGE followed by immunoblot analysis with the indicated antibodies. Filters were exposed for 30 s. (C and D) HCV RNA copy number (C) and titer (D) in the medium were determined as described in Materials and Methods. Values from three measurements in the same experiment are shown, each denoted by an individual dot. Asterisks denote the level of statistical significance (Student's test) between GFP siRNA and apoB siRNA-transfected cells. ∗, P < 0.005. Similar results were obtained in one other independent experiment.