Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection

Abstract

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.

FIG. 1.

Role of HCV-associated cholesterol in infection. (A) Effect of cholesterol depletion on HCV infectivity. HCVcc particles (∼2 fmol of the core protein) were treated with B-CD at 0.1, 1, and 5 mg/ml for 1 h at 37°C. After removal of B-CD, Huh-7 cells were infected with the treated virus particles, after which the core protein content of infected cells at 72 h p.i. was determined as an indicator of infectivity, as previously established (24). (B) Effect of cholesterol replenishment on infectivity. After treatment with 5 mg/ml B-CD, virus was treated either with medium alone or with medium containing exogenous cholesterol for 1 h at 37°C. (C) Effect of cholesterol depletion and replenishment on density gradient profiles of the viral particles. The HCVcc treated with 5 mg/ml B-CD was replenished with exogenous cholesterol (1 mM) and then separated by 10-to-60% sucrose gradient ultracentrifugation. The core protein in each fraction was measured. The density of each fraction was determined by refractive index measurement. (D) Effects of cholesterol depletion and replenishment on viral infectivity. Each fraction (see panel C) was infected, and then the core proteins in the cells were measured at 72 h p.i. (E) Effect of cholesterol depletion on the infectivity of HCVpv (genotype 1a) (shaded bars) or the control, VSVdelG-GFP/G (solid bars). The viruses were preincubated with B-CD for 1 h at 37°C before infection. (F) (Left) The culture medium from HCVcc-producing cells was fractionated as described above. For each fraction, the amounts of core and intracellular core (infectivity) are plotted. Peaks of the core (arrow) and infectivity (arrowhead) are indicated. (Center) An aliquot of fraction 8 (peak of the core) was treated with 1 mM cholesterol for 1 h at 37°C. The resultant aliquot and an untreated aliquot of the fraction were subjected to sucrose gradient ultracentrifugation. The core in each fraction was plotted. (Right) The infectivities of fractions (Fr.) 6 and 8 (see the left panel) with or without cholesterol treatment were determined as shown above. Data are means from four independent experiments. Error bars, standard deviations.

FIG. 2.

Effect of SM hydrolysis on viral infectivity. (A) Effect on the infectivity of HCVcc. HCVcc was treated with 0.1, 1, or 10 U/ml SMase for 1 h at 37°C, after which SMase was removed by ultracentifugation. Huh-7 cells were infected with the treated virus, and the core protein content of infected cells was determined at 72 h p.i. (B) Effect on the infectivity of HCVpv (genotype 1a) (shaded bars) or the control, VSVdelG-GFP/G (VSV cont) (solid bars). The viruses were preincubated with SMase for 1 h at 37°C before infection. Data are means from four independent experiments. Error bars, standard deviations.

FIG. 3.

Effects of B-CD or SMase on virus attachment and internalization. (A) Virus attachment to Huh-7 cells was determined at 4°C after treatment of HCVcc with B-CD (1 or 5 mg/ml) or SMase (1 or 10 U/ml). An antibody (Ab) against CD81 was used, in order to ensure that the antibody did not inhibit HCVcc binding (7, 33). Heparinase was used to reduce HCV attachment to the cell. Viral RNA copies were normalized to total cellular RNA, and the normalized RNA copies in the mock-treated sample (−) were arbitrarily set at 100%. (B) Virus internalization was measured in Huh7-25, a CD81-negative subclone (CD81⁻) (3), and Huh7-25-CD81, which stably expresses CD81 (CD81⁺), after treatment of the virions with B-CD or SMase. After internalization for 2 h at 37°C, cells were exposed to trypsin (trypsin +) or phosphate-buffered saline (trypsin −). Huh7-25 was used to ensure that surface-bound virus would be removed by trypsin treatment. The amounts of HCV RNA in Huh7-25 and Huh7-25-CD81 cells infected with untreated HCVcc were assigned the arbitrary value of 100%, respectively. Results are representative of four independent experiments.

FIG. 4.

Compartmentation of HCV structural proteins within DRM fractions. Lysates of HCVcc-infected cells were either treated with 1% TX-100, either on ice or at 37°C, or left untreated, followed by sucrose gradient centrifugation. (A and B) For each fraction, the amount of core protein was determined by an enzyme-linked immunosorbent assay (A), and E1, calnexin, and caveolin-2 were analyzed by Western blotting (B). The amount of core protein in each lysate (TX-100, 37°C; TX-100, 4°C; Untreated) was assigned the arbitrary value of 100%. M, membrane; NM, nonmembrane; DS, detergent soluble. (C) Lysates of 293T cells expressing HCV E1 or E2 protein were either treated with 1% TX-100, either on ice or at 37°C, or left untreated, followed by discontinuous sucrose gradient centrifugation. Each fraction was concentrated in a Centricon YM-30 filter unit and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against calnexin, caveolin-2, Myc (E1), or FLAG (E2). (D) (Top) Structures of HCV envelope genes used. Amino acid positions of HCV are indicated. Signal sequence, transmembrane (TM), and cytoplasmic tail (CT) domains of VSV G protein are shown. (Bottom) Cell lysates expressing chimeric HCV E1 or E2 protein were treated with 1% TX-100 on ice or left untreated, followed by discontinuous sucrose gradient centrifugation. It has been reported that VSV-G is not associated with lipid (39). Calnexin, caveolin-2, and chimeric glycoproteins (chimeric E1 and chimeric E2) were analyzed by immunoblotting. Fractions are numbered from 1 to 9 in order from top to bottom (light to heavy).

FIG. 5.

Effects of B-CD or SMase treatment of cells on HCV infectivity. Huh-7 cells were either left untreated or treated with B-CD at 0.1, 1, or 5 mg/ml (A) or with SMase at 0.1, 1, or 10 U/ml (B) prior to HCVcc infection. Intracellular core levels were quantitated 72 h p.i. Data are means from four independent experiments. Error bars, standard deviations.

FIG. 6.

Anti-HCV effects of inhibitors of the sphingolipid biosynthetic pathway. Subgenomic replicon cells derived from HCV isolate N or JFH-1, as well as HCVcc-producing cells, were treated with ISP-1 (0.1, 1, or 10 μM), HPA-12 (0.1, 1, or 10 μM) or alpha interferon (IFN) (100 U/ml) for 72 h. HCV RNA titers in the replicon cells (A) and the HCV core protein content of the culture medium of infected cells (C) were determined. Data are means from four independent experiments. Error bars, standard deviations. (B) De novo synthesis of sphingolipid in the absence or presence of ISP-1 (10 μM) and HPA-12 (10 μM) was monitored in duplicate by metabolic labeling with [¹⁴C]serine for 2 h at 37°C. Cer, ceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine.