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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Hepatitis C virus (HCV) relies on host lipid metabolic pathways for its replication, assembly, secretion, and entry. HCV induces de novo lipogenesis, inhibits β-oxidation, and lipoprotein export resulting in a lipid-enriched cellular environment critical for its proliferation. We investigated the effects of a hypolipidemic agent, nordihydroguaiaretic acid (NDGA), on host lipid/fatty acid synthesis and HCV life cycle. NDGA negated the HCV-induced alteration of host lipid homeostasis. NDGA decreased sterol regulatory element binding protein (SREBP) activation and enhanced expression of genes involved in β-oxidation. NDGA inhibited very low-density lipoprotein (VLDL) secretion by affecting mediators of VLDL biosynthesis. Lipid droplets (LDs), the neutral lipid storage organelles, play a key role in HCV morphogenesis. HCV induces accumulation and perinuclear distribution of LDs, whereas NDGA most notably reduced the overall number and increased the average size of LDs. The antiviral effects of NDGA resulted in reduced HCV replication and secretion. Conclusion: NDGA-mediated alterations of host lipid metabolism, LD morphology, and VLDL transport appear to negatively influence HCV proliferation. (HEPATOLOGY 2011;)

Hepatitis C virus (HCV) infection represents a global pandemic with an estimated 3% of world population infected.1 Chronic HCV infection results in severe liver diseases such as steatosis, cirrhosis, and hepatocellular carcinoma.1, 2 HCV is a single-stranded positive sense RNA virus of the Flaviviridae family. HCV is critically dependent on host lipid metabolism for its proliferation.3 Lipidomic analysis during acute HCV infection identified temporal fluctuations in specific lipid species (e.g., phospholipids and sphingomyelins) that play key roles in viral replication, virion morphogenesis/assembly, and secretion.4 HCV is shown to deregulate lipid homeostasis by modulating key players such as sterol regulatory element binding proteins (SREBPs), peroxisome proliferator-activated receptor alpha (PPARα), and carnitine palmitoyl acyl-CoA transferase 1a (CPT1a), resulting in increased lipogenesis, lipid uptake, and reduced β-oxidation, creating a lipid-rich microenvironment conducive to viral proliferation.5-8 Inhibiting SREBP activation, fatty acid synthase (FAS) activity or cholesterol biosynthesis abrogates HCV replication.9-11

HCV replication occurs in close proximity to lipid droplets (LDs) and this proximity facilitates the downstream viral morphogenetic events.12, 13 HCV infection induces accumulation and redistribution of LDs around the perinuclear region.14 Several reports link HCV secretion to cellular very low-density lipoprotein (VLDL) secretion. Silencing apolipoprotein B-100 (ApoB) or pharmacological inhibition of microsomal triglyceride transfer protein (MTP) inhibits HCV secretion.15, 16 ApoE is also shown to be involved in HCV entry/infectivity and virion morphogenesis.17, 18 HCV infection causes a decline in VLDL secretion19, 20 and correlates with hypobetalipoproteinemia seen in chronic HCV patients.21 These observations strongly support the role of VLDL during HCV secretion and highlight the importance of intracellular lipid enriched microenvironment and lipoprotein export for HCV proliferation. Reagents/bioactive molecules that can perturb the viral interference on cellular lipid homeostasis can potentially hamper the viral infectious process.

Nordihydroguaiaretic acid (NDGA) {l,4-bis (3,4-dihydroxyphenyl) 2,3-dimethyl-butane} is a phenolic antioxidant derived from the desert shrub Larrea tridentata.22 It has antioxidant, antiinflammatory, and antiproliferative properties and has been used as traditional medicine against diabetes and other health problems.22 NDGA was used to stabilize hepatic lipid deregulation in high-fat-diet-fed mice by improving lipid breakdown.23, 24 The hypolipidemic effects of NDGA prompted this investigation to determine its influence on HCV infection. Here we report the effect of NDGA on various aspects of the HCV lifecycle eventually leading to inhibition of viral proliferation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Reagents and Plasmids.

NDGA and BSA-conjugated oleic acid were from Sigma-Aldrich (St. Louis, MO). Antibodies against actin, PPARα, and FAS were from Santa Cruz Biotechnology (Santa Cruz, CA). Sources of other antibodies used: anti-ApoB (Chemicon International, Temecula, CA), anti-SREBP-1 and anti-LC3 (Abcam, Cambridge, MA), anti-HCV core, (Affinity BioReagents, Golden, CO). Monoclonal anti-HCV-NS5A and JC1-p7-RLuc2A plasmid25 were gifts from Dr. C. Rice (Rockefeller University, NY). Anti-ApoE was a gift from Dr. G. Luo (University of Kentucky, Louisville, KY). Reporter plasmids SRE-Luc, FAS-Luc, LDLr-Luc, MTP-Luc, and HCV-5′ NCR-Luc plasmids have been described.5, 20, 26 JFH1 clone was a gift from Dr. T. Wakita (Tokyo, Japan). The genotype 2a (JFH1) full-length (FL-Feo) and subgenomic replicon and genotype 1b subgenomic replicon (BM45-Feo) plasmids containing neomycin-luciferase fusions were gifts from Dr. David Wyles (University of California, San Diego).

Cell Culture, HCV Infection, and Foci Forming Unit (FFU) Assay.

The Huh7 subgenomic or full-length JFH replicon cells were maintained in 500 μg/mL Geneticin. The Huh7.5.1 cell line was a gift from Dr. Frank Chisari (Scripps Institute, La Jolla, CA). NDGA was dissolved in dimethyl sulfoxide (DMSO) and added to culture medium with DMSO concentration not exceeding 0.1%. The cytotoxicity of NDGA was determined at 48 hours using the TOX-8 cytotoxicity kit (Sigma-Aldrich), per the manufacturer's instructions. In vitro-synthesized viral genomic RNAs (RiboMax RNA production system, Promega, Madison, WI) were electroporated into Huh7.5.1 cells using cytomix buffer as described.27 The cells were passaged at confluency every 2 to 3 days and culture supernatants collected. The infectivity of the supernatant was determined by foci forming unit (FFU) assays as described.28 Huh7 cells infected with JFH virus (Huh7-JFH) at 0.01 multiplicity of infectivity (MOI) were used for experiments 9-12 days postinfection. Huh7.5.1 cells infected with JCI or JC1p7-RLuc2A virus at 0.01 MOI were used 5 days postinfection. The relative levels of JC1p7-RLuc2A reporter virions in culture supernatants were analyzed by infecting naïve Huh7.5.1 cells and measuring the transduced renilla luciferase activity 72 hours postinfection as described.25 Intracellular infectivity was determined as described15 and intracellular/extracellular HCV RNA was quantified by real-time polymerase chain reaction (PCR) as described.29 HCV core enzyme-linked immunosorbent assay (ELISA) was done using the ORTHO HCV antigen ELISA kit (Wako Chemicals, Richmond, VA), per the manufacturer's instructions. HCV pseudoparticle assays for analyzing HCV entry were carried out as described.30

Quantitative Real-Time PCR.

The relative messenger RNA (mRNA) levels were quantified by comparative CT method by subjecting the complementary DNA (cDNA) to real-time quantitative PCR (qPCR) using DyNAmo SYBR qRT-PCR mix (Finnzymes, Finland), per the manufacturer's instructions. Gene-specific primers used are detailed in the Supporting Information.

Immunofluorescence.

Fluorescent immunostaining was performed as described.27 For staining lipid droplets 1 μg/mL of Bodipy-493/503 was used. The images were analyzed with an Olympus FluoView 1000 confocal microscope.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

NDGA Inhibits HCV Replication.

NDGA induces hypolipidemic effects by altering host lipid metabolism.23, 24 We investigated the NDGA-mediated hypolipidemic actions on HCV-induced lipid homeostasis and its effect on the viral infectious process. Initially to evaluate cytotoxicity, Huh7 cells were treated with various concentrations (10-100 μM) of NDGA for 48 hours. NDGA had a dose-dependent effect on Huh7 cell viability with a CC50 value of 70 μM (Supporting Fig. S1A). Huh7 cells harboring full-length replicon (FL-Feo) of HCV genotype-2a with an in-frame luciferase gene were treated with various concentrations of NDGA for 48 hours and HCV replication assessed by luciferase activity. NDGA showed a dose-dependent inhibition of HCV replication, with a median effective concentration (EC50) value of about 30μM (Fig. S1B). For subsequent studies, 35 μM NDGA was used. We then analyzed the effect of 35 μM NDGA on replication of subgenomic and full-length HCV-JFH replicons at 24 hours and 48 hours posttreatment. In both the subgenomic and full-length replicon cells the luciferase activity in untreated control cells increased from 24 hours to 48 hours but decreased by about 70% after 48 hours in NDGA-treated cells (Fig. 1A). The HCV-JFH cell culture-based infection system (HCVcc) also yielded similar results. At 48 hours after NDGA treatment the intracellular levels of HCV RNA in infected cells was suppressed by about 70% compared to untreated cells (Fig. 1B). In correlation with the above results, the immunoblot analysis of HCV proteins after 24 hours and 48 hours NDGA treatment in replicon and infected cells also showed a similar decline in the HCV protein levels compared to untreated controls (Fig. 1C). We also analyzed the effect of NDGA on genotype 1b subgenomic replicon cells (BM45-Feo). Genotype 1b replication was decreased by about 60% after 48 hours of NDGA treatment (Fig. S2), indicating its genotype-independent effects on viral replication. Together, these results indicate that NDGA inhibited both the HCV RNA and protein expression.

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Figure 1. NDGA inhibits HCV replication. Huh7-JFH replicon cells harboring full-length or subgenomic HCV genome of genotype 2a (JFH) and Huh7 cells persistently infected with culture derived HCV-JFH virus were treated with 35 μM NDGA for the indicated time. The control cells were treated with DMSO. (A) HCV replication in the replicon cells was determined as a function of the firefly luciferase activity. (B) HCV replication in JFH-infected cells determined by qRT-PCR of intracellular HCV RNA and data shown as genome equivalents (GE) of HCV RNA per μg total RNA. The experiments were carried out in triplicate and data shown are mean ± SEM. (C) Western blot analysis showing indicated HCV proteins in the full-length genotype 2a JFH replicon (FL-Feo) and infected (JFH) cell lysates after NDGA treatment. β-Actin was used as protein loading control.

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We next investigated if NDGA treatment also affects HCV IRES-dependent translation. Huh7 cells pretreated with NDGA for 24 hours were electroporated with replication defective GND mutant (JFH-GND) HCV RNA and continued to be treated with NDGA. The GND mutant RNA is defective in replication but is translated efficiently. Cell lysates were analyzed for HCV protein expression by western blot at 8 hours postelectroporation. We observed no significant difference in levels of HCV Core and NS5A proteins in NDGA pretreated or untreated cells (Fig. 2A). The intracellular levels of GND mutant RNA was determined by qRT-PCR at 3 hours, 8 hours, 16 hours, and 24 hours postelectroporation in both NDGA-treated and untreated cells to determine the electroporation efficiency and RNA decay. The results show that NDGA does not affect RNA stability (Fig. 2B). Overall, these results suggest that NDGA treatment does not hinder HCV RNA translation or mediate its degradation. The effect of NDGA on HCV-IRES translation was also analyzed using luciferase reporter plasmid HCV-5′NCR-Luc.26 No significant variation in the HCV-IRES luciferase reporter activity was observed (Fig. 2C). These results suggest that the inhibition in HCV RNA replication and protein expression caused by NDGA is not a consequence of inhibition in HCV IRES-mediated translation.

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Figure 2. NDGA does not affect HCV-IRES translation. Huh7 cells pretreated with NDGA or DMSO treated for 48 hours were electroporated with replication defective JFH-GND RNA and cell lysates analyzed for HCV protein expression 8 hours postelectroporation. (A) Western blot analysis showing expression of HCV core and NS5A proteins. Cell lysates of persistently infected cells (Huh7-JFH) were used as positive control and β-actin as protein loading control. (B) Intracellular levels of JFH-GND RNA in respective cell lysates at 3 hours, 8 hours, 16 hours, and 24 hours postelectroporation determined by qRT-PCR analysis and data shown as genome equivalents (GE) of HCV RNA per μg total RNA. (C) The HCV-IRES activity determined by HCV-IRES luciferase reporter plasmid HCV 5′NCR-Luc. Huh7 cells pretreated with NDGA or DMSO for 24 hours were transfected with HCV 5′NCR-Luc RNA and treatment with NDGA or DMSO continued for a further 24 hours prior to estimation of firefly luciferase activity in cell lysates. Renilla luciferase plasmid was used as transfection control.

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NDGA Subverts HCV-Induced Alterations of Lipogenesis.

HCV gene expression stimulates lipogenesis.3 HCV activates master regulators of cholesterol/lipid biosynthetic enzymes, SREBP-1 and -2, and reduces expression of PPARα and CPT1a.5-8 To determine the effect of NDGA on SREBP activation, HCV-infected and uninfected Huh7 cells were transfected with SRE-luciferase reporter plasmids and treated with NDGA for 48 hours posttransfection. Luciferase activity was reduced by about 60% in both the infected and uninfected NDGA-treated cells (Fig. 3A). Further, we used luciferase reporter plasmids in which promoters of FAS or low-density lipoprotein receptor (LDLr) genes control transcription. In both cases, 48-hour NDGA treatment resulted in about a 60% decline in luciferase activity compared to untreated controls (Fig. 3A). In contrast, HCV infection stimulated the transcription of SREBPs, FAS, and LDLr (Fig. 3A, see cont Huh7 vs. cont JFH-Huh7). Both FAS and LDLr promoters contain SREBP binding sites and are upregulated by SREBPs in addition to other transcription factors.31, 32 We then evaluated variations in the mRNA levels of a few representative genes such as FAS, SREBP1c, SREBP2, PPARα, and CPT1a in HCV-infected and uninfected Huh7 cells treated with and without NDGA for 48 hours. Consistent with previous reports, HCV infection increased levels of FAS mRNA and reduced PPARα and CPT1a mRNA (Fig. 3B). In contrast, 48-hour NDGA treatment subverted HCV lipogenic signaling by increasing PPARα and CPT1a mRNA levels and reducing FAS mRNA (Fig. 3B). Immunoblot analysis of FAS, PPARα, and SREBP-1 proteins in HCV infected and uninfected Huh7 cells treated with or without NDGA further support the data in Fig. 3B (Fig. 3C). Overall, these results suggest that NDGA-mediated inhibition of HCV replication may be a consequence of its effects on lipid metabolism. To evaluate this hypothesis, NDGA-treated cells were supplemented with oleic acid (100 μM). Oleic acid supplementation partially restored HCV replication in NDGA-treated cells (Fig. S3A). The multiple effects of NDGA on lipid metabolism may be the reason for incomplete reversal of NDGA effect on replication by oleic acid (Fig. S3A).

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Figure 3. NDGA subverts the HCV-altered/induced lipid biosynthetic proteins. Uninfected (Huh7) or persistently infected (Huh7-JFH) cells were transiently transfected with the respective reporter plasmids and treated with NDGA or DMSO for 48 hours. The cell lysate was then used to determine firefly luciferase activity. Renilla luciferase plasmid was used as transfection control. (A) The firefly luciferase activity of the indicated reporter plasmids normalized against renilla luciferase activity after NDGA or DMSO treatment in Huh7 and HCV-infected Huh7 (Huh7-JFH) cells. (B) Alterations in mRNA levels of the indicated genes determined by qRT-PCR as described in Materials and Methods in NDGA or DMSO-treated Huh7 and persistently infected Huh7-JFH cells relative to the DMSO-treated Huh7 cells. (C) Western blot analysis of the lysate used for experiment described in (B) for indicated proteins. All experiments were carried out in triplicate and data shown are mean ± SEM.

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NDGA Reduces the Number and Enhances Size of LDs.

Alteration of host lipid metabolism associated with HCV infection leads to accumulation of LDs.2, 3, 13, 14 Our studies thus far showed that NDGA subverted HCV-mediated alterations of lipid metabolism by preventing SREBP activation and inducing genes involved in lipid breakdown. Hence, we studied the effect of NDGA on accumulation and arrangements of LDs in HCV-infected cells. Correlating with previous reports,14 we observed that HCV infection led to significant induction and rearrangement of LDs around the perinuclear space, in contrast to uninfected cells (Fig. 4A; see HCV-infected cells marked with * sign vs. uninfected cells marked with + sign in the merged panel). Under normal physiologic conditions, Huh7 cells display few LDs (see Huh7 cells vs. HCV-infected or oleic acid-stimulated Huh7 cells in Fig. 4B). In oleic acid-stimulated or HCV-infected Huh7 cells, NDGA treatment led to a decrease in number and increase in average size of the LDs (Fig. 4B,C,D). The increase in size and reduction in number of LDs caused by NDGA treatment (Fig. 4C,D) could be due to fusion activity between LDs and/or delivery of newly synthesized lipids to preexisting LD.33, 34 It has been shown that lipophagy regulates intracellular lipid stores.35 Further investigations on possible induction of lipophagy by NDGA did not reveal any lipophagy being induced by NDGA treatment (Fig. S4A), as there was no colocalization of LDs with the lysosomal protein Lamp1 or enhanced lipidation of LC3-I to LC3-II, although the overall levels of LC3 protein increased (Fig. S4B).

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Figure 4. NDGA severely affects lipid droplet numbers and morphology. (A) HCV-infected Huh7-cells (Huh7-JFH) were stained for LDs using Bodipy and HCV core protein and imaged as described in Materials and Methods. The LDs are shown in green and HCV core protein in Red. The * and + signs in the merged panel indicate HCV-infected and uninfected Huh7 cells, respectively. (B) Control Huh7, oleic acid stimulated Huh7, and HCV (JFH)-infected Huh7 cells were treated with NDGA or DMSO for 48 hours and stained for LDs. Upper row represents Huh7 cells treated with DMSO or NDGA. Middle row shows Huh7 cells supplemented with 100 μM BSA-oleic acid complex and treated with DMSO or NDGA. The lower row displays HCV-infected (JFH) Huh7 cells treated with DMSO or NDGA. (C) Relative quantitation of change in LDs size and (D) relative quantitation of change in LDs number, number in NDGA or DMSO-treated HCV (JFH)-infected Huh7 cells. Quantitation was done manually in 20 individual cell images from NDGA-treated and untreated conditions using ImageJ software.

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NDGA Suppresses HCV Assembly and Secretion.

HCV replication occurs in close juxtaposition to LDs and the viral particle morphogenesis requires interaction of core and NS5A proteins with LDs.12, 13 Because NDGA treatment altered the distribution, number, and morphology of LDs, we investigated if NDGA treatment affects viral assembly and secretion. The intracellular infectivity of JCI infected Huh7.5.1 cells treated with and without NDGA for 48 hours was evaluated as described.15 The results indicate that NDGA treatment causes a reduction in accumulation of matured infectious viral particles compared to untreated cells (Fig. 5C). We assumed that increasing the LD number restores the HCV assembly defect observed in NDGA-treated cells. Oleic acid supplementation to NDGA-treated cells partially restored HCV intracellular infectivity, although not to the level seen in controls (Fig. S3B). Our earlier results show that oleic acid supplementation in NDGA-treated cells did not increase the LD number as dramatically as triggered by oleic acid alone (Fig. 4B). Hence, the marginal increase in LD number mediated by oleic acid stimulation of NDGA-treated cells only contributed to a modest rise in intracellular infectivity (Fig. S3B).

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Figure 5. NDGA inhibits HCV secretion. (A) Persistently infected Huh7.5.1 cells were treated with NDGA or DMSO and the accumulation of HCV viral particles in the culture supernatant evaluated at indicated timepoints by qRT-PCR analysis of HCV RNA isolated from culture supernatant and data shown as genome equivalents (GE) of HCV RNA per mL culture supernatant. (B) The level of HCV core protein in the 48-hour culture supernatants used in experiment (A) were analyzed by OrthoHCV core antigen ELISA kit as described in Materials and Methods. (C) Infectivity of the 48-hour culture supernatants used in experiment (A) (extracellular infectivity) and intracellular infectivity of crude lysates of cells used in experiment (A) was determined by FFU assay as described in Materials and Methods. (D) Inhibition in HCV secretion by NDGA assayed using the JCI-p7-Rluc 2A reporter virus. Infectivity of the culture supernatants was determined by estimating the renilla luciferase activity 72 hours postinfection as described in Materials and Methods. The naïve Huh7.5.1 cells were infected with samples of culture supernatants undiluted or those at various dilutions, collected 48 hours post-NDGA or DMSO treatment as indicated. Intracellular renilla luciferase activity in lysates of parent cultures was used to determine the effect on HCV replication. All the experiments were carried out in triplicate and the results shown are mean ± SEM.

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We then evaluated the secretion of viral particles into culture supernatants in JCI-infected Huh7.5.1 cells treated with NDGA for 48 hours. Analysis of extracellular HCV RNA purified from infectious culture supernatants showed that 48-hour NDGA treatment led to a 2 log-fold reduction in the HCV secretion compared to untreated control (Fig. 5A). Analysis of HCV core protein levels in culture supernatant by ELISA also yielded close to 2 log-fold declines in level of core protein after 48 hours NDGA treatment (Fig. 5B). The FFU assay, a more reliable functional assay for analyzing infectivity of infectious HCV culture supernatants, also confirmed that the supernatants of NDGA-treated cultures were about 2 log-fold less infectious compared to untreated control (Fig. 5C). We also performed a similar functional assay using the JCI-p7Rluc2A reporter virus.25 Huh7.5.1 cell cultures infected with JCI-p7Rluc2A reporter virus were treated with or without NDGA for 48 hours and naïve Huh7.5.1 cells were infected with log fold dilutions of supernatants of untreated control cultures and with undiluted supernatants from NDGA-treated cultures, respectively. The infectivity was determined by assaying for transduced renilla luciferase activity in cell lysates 72 hours postinfection (Fig. 5D). The results indicate that the infectivity of undiluted supernatant from NDGA-treated cultures was close to the infectivity of 400-fold diluted untreated culture supernatant (Fig. 5D). The level of HCV RNA replication in the NDGA-treated and untreated parent cultures was also evaluated by estimating intracellular renilla luciferase activity (Fig. 5D). The reference lines drawn on the bar graph depict the log fold difference between HCV replication and secretion levels in NDGA-treated and untreated samples. These results indicate that NDGA inhibits HCV replication and assembly by ∼60% but more strongly effects HCV secretion by 2 log-fold (Fig. 5D). Next we analyzed the localization of core and NS5A on LDs after NDGA treatment by immunofluorescence (Fig. S5). In agreement with earlier results on HCV replication, NDGA treatment resulted in diminution of core and NS5A protein levels and perturbed the typical perinuclear distribution pattern observed in untreated infected cells for both the proteins (Fig. S5). However, the proteins still localized to the periphery of LDs, suggesting that NDGA treatment did not interfere with their interaction to LDs but affects their expression and cytosolic distribution.

NDGA Inhibits VLDL Secretion.

It is widely acknowledged that HCV co-opts the VLDL secretory pathway for its egress.15, 16, 36, 37 MTP protein initiates VLDL biosynthesis in endoplasmic reticulum (ER) by cotranslational lipidation of nascent ApoB in the ER.38 Because NDGA treatment reduced the secretion of virus (Fig. 5), we investigated if NDGA inhibited MTP expression. Huh7 cells transfected with MTP-promoter reporter plasmid were treated with NDGA for 48 hours. NDGA treatment led to a 40% reduction of MTP promoter activity (Fig. 6A), indicating that NDGA inhibits MTP gene transcription. Analysis of MTP mRNA levels also yielded similar results (Fig. 6B). Unlipidated ApoB is subjected to proteasomal degradation39; in correlation with this we observed a decline in the intracellular levels of ApoB in NDGA-treated cells (Fig. 6C), although the ApoB mRNA levels did not significantly alter (Fig. 6B), indicating an inhibition of MTP activity by NDGA. Inhibition in VLDL synthesis after MTP-mediated ApoB lipidation results in accumulation of lipidated ApoB as crescents on the periphery of the LDs.40 Analysis of cytoplasmic distribution of ApoB in NDGA-treated and untreated Huh7 cells by immunofluorescence revealed a characteristic reticular distribution pattern of ApoB in control cells but enhanced number of ApoB crescents on periphery of LDs in NDGA-treated cells (Fig. 6D). This suggests that NDGA treatment results in the inhibition of VLDL biosynthesis downstream of MTP-mediated lipidation of ApoB. Recently, proteins such as acyl-CoA synthetase 3 (ACSL3) and phospholipid transfer protein 1 (PLTP1) have been shown to play a crucial role in VLDL biosynthesis and secretion downstream of MTP.37, 41 Analysis of mRNA levels of these proteins after NDGA treatment resulted in about 4-fold reduction in ACSL3 mRNA and nearly 2-fold reduction in PLTP1 mRNA (Fig. 6B). These observations suggest that NDGA inhibits VLDL secretion by inhibiting MTP expression and activity and also events downstream of MTP. We examined the secretion of VLDL particles by directly monitoring the levels of ApoB and ApoE proteins in the culture supernatants, as described.16 NDGA treatment completely abolished VLDL secretion in both uninfected and HCV-infected Huh7 cells (Fig. 6C). Correlating with this result, we observed that oleic acid supplementation did not restore HCV secretion in NDGA-treated cells (Fig. S3C). Oleic acid supplementation does restore lipid levels but could not revert NDGA-mediated transcriptional reprogramming, and hence cannot restore HCV secretion in NDGA-treated cells. Interestingly, NDGA also completely abolished secretion of α1-antitrypsin, suggesting that it inhibited general intracellular secretory transport (Fig. 6C). In contrast to ApoB, intracellular levels of ApoE increased with NDGA treatment (Fig. 6C) probably due to the accumulation of unsecreted ApoE and not enhanced expression because we observed that ApoE mRNA levels remain constant in NDGA-treated and untreated cells (Fig. S6). Overall, the results suggest that NDGA inhibits VLDL secretion by inhibition of MTP activity and down-regulation of genes essential for VLDL secretion pathway.

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Figure 6. NDGA inhibits VLDL secretion. (A) Effect of NDGA on MTP promoter activity. To determine the MTP promoter activity Huh7 cells were transiently transfected with MTP-promoter-Luc reporter plasmid followed by treatment with NDGA or DMSO for 48 hours. The firefly luciferase activity in cell lysates was then determined to estimate the relative MTP promoter activity. The values were normalized against the renilla luciferase activity, which was used as transfection control. (B) Changes in the mRNA levels of indicated genes in NDGA-treated Huh7 cells relative to DMSO-treated cells were determined by qRT-PCR as described in Materials and Methods. All experiments were carried out in triplicate and the results shown here are mean ± SEM. (C) Intracellular levels of ApoB and ApoE were determined by western analysis of uninfected Huh7 and infected (JFH) cells treated with NDGA or DMSO for 48 hours. VLDL secretion in the culture supernatants of above experiment was determined by western blot analysis of ApoB and ApoE levels in culture supernatants. (D) Immunofluorescence of ApoB in NDGA-treated or untreated Huh7 cells. ApoB is shown in red and LDs in green and the nuclei counterstained with DAPI in blue.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Targeting host factors that facilitate viral proliferation can be an effective strategy in designing specific antiviral agents. The various stages of HCV viral life cycle such as entry, replication, and viral assembly and/or secretion are dependent on cellular lipid metabolism.3, 9, 15 Here we explored the hypolipidemic effects of NDGA on the HCV life cycle. HCV infection generates a lipid-rich microenvironment conducive for its proliferation by deregulating lipid homeostasis.5-8, 19, 20 Here we show that NDGA subverts HCV-induced alterations of lipid homeostasis and thwarts cellular lipid enrichment induced by HCV. NDGA reduced the activation of SREBPs and their target genes FAS and LDLr (Fig. 3A,B). Unlike HCV infection, NDGA enhanced the expression of PPARα and its downstream target CPT1a (Fig. 3B), implying that NDGA decreases hepatic lipogenesis and enhances catabolic breakdown of lipids. NDGA prevented fatty liver in high-fat-fed mice by up-regulating the adenosine monophosphase (AMP)-activated protein kinase (AMPK), a central player in lipid and glucose homeostasis.24 Interestingly, HCV infection has been shown to inhibit AMPK activation by way of protein kinase B and activation of AMPK abrogated lipid accumulation and inhibited HCV replication.42 HCV infection induces modified membranous structures, on which it assembles its replication complexes that engage in RNA replication activities.43 Such membrane alterations require enrichment and reorganization of specific lipids and lipid-protein interactions.10, 44 NDGA through its hypolipidemic actions can alter such effects and inhibit HCV replication. NDGA treatment of persistently infected or replicon cells that possess well-established replication complexes resulted in a modest decline in replication, whereas the inhibition was more profound in cells in which infection was yet to be established (data not shown).

The most notable effect of NDGA was on the LDs. NDGA-treated HCV infected cells displayed a reduced number and enlarged LDs (Fig. 4B). The LDs are enclosed by phospholipid monolayers, and when phospholipids are limiting, fusion of LDs is induced to overcome the reduced surface-to-volume ratio of droplets.34 However, the mechanism behind the observed increase in the size of LDs after NDGA treatment is not clearly understood. HCV infection results in induction and relocation of LDs, which play a key role in virion morphogenesis.12-14 Carboxyl-esterase 1 (CES1), an enzyme that regulates LDs abundance and size, has recently been shown to be activated during HCV infection and positively influence HCV secretion.45 NDGA treatment reduced intracellular infectivity of infected cells (Fig. 5C), although NDGA did not influence the interaction of HCV core or NS5A to LDs (Fig. S4), suggesting that the negative influence of NDGA on virion assembly is a consequence of alteration of numbers of LDs. NDGA reduces HCV replication by about 60%-70% and this can lead to reduced intracellular infectivity. However, the magnitude of NDGA-mediated reduction in HCV secretion (∼2 log fold) is greater than HCV replication (∼60%-70%). This should cause an accumulation of virions in NDGA-treated cells. However, we observed less intracellular infectivity compared to controls in NDGA-treated cells, suggesting that NDGA also inhibits HCV virion assembly in addition to inhibiting replication and secretion. Numerous studies support the notion that HCV exploits VLDL secretion for egress.15, 16, 36 NDGA treatment abrogated ApoB/VLDL secretion (Fig. 6) by way of reduction in MTP transcript levels (Fig. 6A,B). In addition, NDGA also affects MTP activity per se, which correlates with the observed decline in intracellular levels of ApoB, although the mRNA levels are not affected (Fig. 6B,C) because unlipidated ApoB has been shown to be subjected to proteasomal degradation.39 We also observed increased instances of ApoB crescents on LD surfaces after NDGA treatment (Fig. 6D). ApoB crescents comprise lipidated ApoB destined for degradation and result from inhibition of VLDL pathway downstream of ApoB lipidation.40 These studies suggest that NDGA downregulates VLDL secretion by affecting MTP expression/activity and by inhibiting subsequent steps downstream of ApoB lipidation, probably by down-regulating PLTP1 and ACSL3 (Fig. 6B), two important genes involved in VLDL assembly. The failure to reverse NDGA effects on HCV secretion by oleic acid suggests that mere enrichment of lipids does not revert all the effects of NDGA, such as transcriptional modulation, trafficking, and other unknown signaling events. Apart from inhibiting VLDL secretion, NDGA also affected the secretion of hepatic secretory proteins such as α1-antitrypsin, indicating a global inhibition of secretory pathway. Indeed, NDGA has been shown to inhibit secretory protein transport by affecting the ER to Golgi transport.46

In summary, our studies show that NDGA, due to its overall effects on host lipid metabolism, can potentially serve to abrogate HCV infection. Developing NDGA derivatives with enhanced specificity, potency, and less cytotoxicity holds promise as an antiviral agent against not only HCV but also other RNA viruses, which rely on host lipid metabolism.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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