Hepatitis C virus stimulates low-density lipoprotein receptor expression to facilitate viral propagation

Abstract

Lipids play a crucial role in multiple aspects of hepatitis C virus (HCV) life cycle. HCV modulates host lipid metabolism to enrich the intracellular milieu with lipids to facilitate its proliferation. However, very little is known about the influence of HCV on lipid uptake from bloodstream. Low-density lipoprotein receptor (LDLR) is involved in uptake of cholesterol rich low-density lipoprotein (LDL) particles from the bloodstream. The association of HCV particles with lipoproteins implicates their role in HCV entry; however, the precise role of LDLR in HCV entry still remains controversial. Here, we investigate the effect of HCV infection on LDLR expression and the underlying mechanism(s) involved. We demonstrate that HCV stimulates LDLR expression in both HCV-infected Huh7 cells and in liver tissue from chronic hepatitis C patients. Fluorescence activated cell sorting and immunofluorescence analysis revealed enhanced cell surface and total expression of LDLR in HCV-infected cells. Increased LDLR expression resulted in the enhanced uptake of lipoprotein particles by HCV-infected cells. Analysis of LDLR gene promoter identified a pivotal role of sterol-regulatory element binding proteins (SREBPs), in the HCV-mediated stimulation of LDLR transcription. In addition, HCV negatively modulated the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that facilitates LDLR degradation. Ectopic expression of wild-type PCSK9 or gain-of-function PCSK9 mutant negatively affected HCV replication. Overall, our results demonstrate that HCV regulates LDLR expression at transcriptional and posttranslational level via SREBPs and PCSK9 to promote lipid uptake and facilitate viral proliferation.

Importance: HCV modulates host lipid metabolism to promote enrichment of lipids in intracellular environment, which are essential in multiple aspects of HCV life cycle. However, very little is known about the influence of HCV on lipid uptake from the bloodstream. LDLR is involved in uptake of cholesterol rich lipid particles from bloodstream. In this study, we investigated the effect of HCV on LDLR expression and the underlying mechanism triggered by the virus to modulate LDLR expression. Our observations suggest that HCV upregulates LDLR expression at both the protein and the transcript levels and that this upregulation likely contributes toward the uptake of serum lipids by infected hepatocytes. Abrogation of HCV-mediated upregulation of LDLR inhibits serum lipid uptake and thereby perturbs HCV replication. Overall, our findings highlight the importance of serum lipid uptake by infected hepatocytes in HCV life cycle.

FIG 1

HCV stimulates LDLR protein expression. (A) Quantitative real-time PCR (qRT-PCR) analysis of LDLR mRNA in mock- and HCV-infected Huh7 cells. The data presented are means ± the standard errors of the mean (SEM; n = 3). Statistical significance was determined by calculating P value using the Student unpaired two-tailed t test (*, P < 0.005). (B) Western blot analysis of LDLR in mock- and HCV-infected Huh7 cells. HCV NS5A and β-actin were used as the infection marker and the protein loading control, respectively. (C) Densitometric analysis of LDLR band shown in panel B, normalized against β-actin. (D) Western blot analysis of LDLR in liver biopsy samples from chronic hepatitis C patients (samples 3 to 7) and non-HCV donors (samples 1 and 2).

FIG 2

HCV stimulates both the surface and total expression of LDLR. (A) Mock- and HCV-infected Huh7 cells stained for cell surface LDLR were subjected to FACS. Blue and red curves represent mock- and HCV-infected cells, respectively. (B) Confocal image of HCV-infected Huh7 cells immunostained for total cellular LDLR (green) and HCV NS5A (red). Nuclei are stained with DAPI (blue). Infected and uninfected cells are demarcated by “+” and “−” signs, respectively. (C) Quantification of LDLR fluorescence intensity in uninfected versus HCV-infected cells. LDLR fluorescence intensity in ∼20 infected and ∼20 uninfected cells from independent images was quantified by ImageJ. The data represent relative fluorescence intensities expressed in arbitrary units as means ± the SEM. P values were calculated using the Student unpaired two-tailed t test (*, P < 0.05).

FIG 3

HCV infection promotes enhanced uptake of Dil-LDL. (A) HCV-infected Huh7 cells were cultured in lipoprotein-deficient serum supplemented with 10 μg of Dil-LDL/ml for 5 h at 37°C, fixed in 4% paraformaldehyde, and immunostained for HCV core, followed by imaging using an FV1000 confocal microscope. HCV core is shown in green and Dil-LDL in red (B). Quantitative analysis of Dil-LDL fluorescence intensity in infected versus uninfected cells. Totals of 100 uninfected and 100 HCV-infected cells from independent images were analyzed using ImageJ. The data represent the relative fluorescence intensities expressed in arbitrary units as means ± the SEM. P values were calculated using the Student unpaired two-tailed t test (*, P < 0.005).

FIG 4

HCV stimulates LDLR gene transcription via SREBPs. (A) As schematic representation of the wild-type and mutational constructs of LDLR promoter luciferase reporters. The schematic representation has been adapted and modified from previous publication (26). (B) Mock- and HCV-infected Huh7 cells were transiently transfected with the respective wild-type and mutational luciferase reporter constructs of LDLR promoter, together with pRSV-β-galactosidase. After 24 h, the luciferase and β-galactosidase activities were determined. The firefly luciferase activity was normalized against β-galactosidase activity to correct for variation in transfection efficiency. The data shown are means ± the SEM (n = 3). P values were calculated using the Student unpaired two-tailed t test, * P < 0.05, ** P < 0.0005.

FIG 5

HCV downregulates PCSK9 at protein level. (A) qRT-PCR analysis of relative levels of PCSK9 mRNA normalized against GAPDH mRNA in mock- and HCV-infected Huh7 cells. The data shown are means ± the SEM of three independent experiments. (B) Western blot analysis of PCSK9 in mock- and HCV-infected Huh7 cells. (C) Schematic representation of deletion and mutational constructs of PCSK9 promoter luciferase reporters. The schematic representation has been adapted and modified from previous publication (27). (D) Mock- and HCV-infected and Huh7 cells were transiently transfected with respective wild-type and mutational luciferase reporter constructs of PCSK9 promoter together with pRSV-β-galactosidase. After 24 h, the luciferase and β-galactosidase activities were determined. The firefly luciferase activity was normalized against the β-galactosidase activity to correct for variations in transfection efficiency. The data shown are means ± the SEM (n = 3). P values were calculated using Student unpaired two-tailed t test (*, P < 0.005; **, P < 0.0005). (E). Analysis of PCSK9 ubiquitination. Mock- and HCV-infected cells were transiently transfected with wild-type PCSK9-Flag and HA-ubiquitin expression vectors. At 36 h posttransfection, the cells were either left untreated or treated with 20 μM MG132 for 5 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by Western blotting with anti-HA antibody. A total of 15% of the total immunoprecipitation input was used for Western blot analysis with anti-HA antibody. (F) Mock- and HCV-infected cells were either left untreated or received 20 μM MG132 treatment for 5 h, and the PCSK9 levels were determined by Western blotting. (G) Western blot analysis of cIAP1 in mock- and HCV-infected Huh7 cells. HCV Core/NS5A was used as an infection marker, and β-actin was used as an internal loading control.

FIG 6

HCV initiates upregulation of LDLR expression at protein level, followed by transcriptional stimulation of LDLR gene. (A) Huh7 cells were either mock infected or infected with HCVcc at an MOI of 5, cells were collected at the indicated time points postinfection, and the LDLR mRNA levels were determined by qRT-PCR of total cellular RNA. LDLR mRNA was normalized against GAPDH mRNA. (B) Huh7 cells transiently transfected with luciferase reporter construct of LDLR promoter, together with pRSV-β-galactosidase, were either mock infected or infected with HCVcc at an MOI of 5. The cells were collected at the indicated time points postinfection, and the firefly luciferase and β-galactosidase activities in the cell lysates were determined as described in ‘Materials and Methods. The luciferase activity was normalized against the β-galactosidase activity to normalize variations in transfection efficiency. The data presented are means ± the SEM (n = 3). P values were calculated by using the Student unpaired two-tailed t test (*, P < 0.05; **, P < 0.005). (C) Western blot analysis of LDLR and PCSK9 in cell lysates obtained from cells described in panel A. HCV NS5A was used as an infection marker, and β-actin was used as a protein loading control. (D and E) Densitometric analysis of the respective LDLR (D) and PCSK9 (E) Western blots shown in panel C. Band intensities were normalized against β-actin bands and are expressed as percentage of the control.

FIG 7

Ectopic expression of wild-type PCSK9 or D374Y mutant negatively affects HCV replication. (A) Full-length replicon cells (FL-Feo cells) of HCV genotype 2a were transiently transfected with wild-type or D374Y mutant PCSK9-Flag expression vector, and at 48 h posttransfection the firefly luciferase activity in cell lysates was determined. (B) At 4 h postinfection, HCVcc-infected Huh7 cells were transfected with wild-type or D374Y mutant PCSK9-Flag expression vector, and at 72 h postinfection the HCV replication was determined by qRT-PCR analysis of HCV RNA. The data presented are means ± the SEM (n = 3). P values were calculated by using a Student unpaired two-tailed t test (*, P < 0.0005). (C) Western blot analysis of LDLR and FLAG-tagged wild-type and mutant PCSK9 in mock- and HCV-infected cells untransfected or transiently transfected with wild-type PCSK9-Flag or D374Y PCSK9-Flag expression vector. HCV NS5A was used as an infection marker, and β-actin was used as protein loading control.