Naringenin inhibits the assembly and long-term production of infectious hepatitis C virus particles through a PPAR-mediated mechanism

Abstract

Background & aims: Hepatitis C virus (HCV) infection affects 3% of the world population and is the leading cause of chronic liver disease worldwide. Current standard of care is effective in only 50% of the patients, poorly tolerated, and associated with significant side effects and viral resistance. Recently, our group and others demonstrated that the HCV lifecycle is critically dependent on host lipid metabolism and that its production is metabolically modulated.

Methods: The JFH1/Huh7.5.1 full lifecycle model of HCV was used to study the antiviral effects of naringenin on viral replication, assembly, and production. Activation of PPARα was elucidated using GAL4-PPARα fusion reporters, PPRE reporters, qRT-PCR, and metabolic studies. Metabolic results were confirmed in primary human hepatocytes.

Results: We demonstrate that the grapefruit flavonoid naringenin dose-dependently inhibits HCV production without affecting intracellular levels of the viral RNA or protein. We show that naringenin blocks the assembly of intracellular infectious viral particles, upstream of viral egress. This antiviral effect is mediated in part by the activation of PPARα, leading to a decrease in VLDL production without causing hepatic lipid accumulation in Huh7.5.1 cells and primary human hepatocytes. Long-term treatment with naringenin leads to a rapid 1.4 log reduction in HCV, similar to 1000U of interferon. During the washout period, HCV levels returned to normal, consistent with our proposed mechanism of action.

Conclusions: The data demonstrates that naringenin is a non-toxic assembly inhibitor of HCV and that other PPARα agonists play a similar role in blocking viral production. The combination of naringenin with STAT-C agents could potentially bring a rapid reduction in HCV levels during the early treatment phase, an outcome associated with sustained virological response.

Fig. 1. Naringenin blocks the assembly of HCV-infectious particles

(A) Naringenin dose-dependently inhibits ApoB-100 and HCV RNA secretion in JFH1-infected Huh7.5.1 cells. HCV RNA did not accumulate in cells. Maximal inhibition of HCV RNA secretion was 74 ± 4% at 200 µM naringenin at 24 h (p < 0.001). (B) 24 h of treatment with 200 µM naringenin decreased the secretion of virus into the media, but did not change the amount of viral RNA or core protein inside cells. Values are normalized to untreated controls. (C) Infected cells were treated for 5 h with 0.1 µg/ml BFA, an inhibitor of Golgi-dependent secretion, 10 µM of BMS-200150, a known MTP inhibitor (MTPi), or with 200 µM naringenin (NN). Infectivity of the secreted virus (extracellular) was quantified by colony titer. Extracellular infectivity was blocked by BFA treatment and inhibited by both BMS-200150 and naringenin, by 45% and 68%, respectively (p < 0.02). (D) To measure changes in the accumulation of intracellular infectious virus particles, the cells were lysed by freeze–thaw cycle and the virus-containing supernatant was used in an infectivity titer assay. BFA treatment led to accumulation of infectious virus in cells. Co-treatment of BFA with BMS-200150 blocked the accumulation of infectious virus in cells, leading to a 46% (p < 0.05) decrease in infectious particle accumulation. Naringenin had a similar effect to that of the MTP inhibitor, leading to a 55% (p < 0.05) decrease in infectious particle accumulation.

Fig. 2. The activity of naringenin is mediated in part by PPARα

(A) Continuous 24 or 96-h treatment with 200 µM naringenin does not cause triglyceride accumulation in primary human hepatocytes. (B) JFH1-infected Huh7.5.1 cells treated with 200 µM naringenin or 10 µM WY14643, a PPARα agonist, show a similar inhibition of MTP activity (p < 0.002), ApoB secretion (p < 0.02), and HCV RNA secretion (p < 0.012), without affecting intracellular levels of HCV core protein (p > 0.50). (C) Chronically infected Huh7.5.1 cells transfected with PPRE reporter plasmid were treated for 24 h with naringenin. Naringenin dose-dependently enhanced PPRE activity by up to 24 ± 7% (p = 0.015). Maximal induction was not different from WY14643 (p = 0.198). (D) HG5LN-PPARα reporter cells in which the PPARα LBD is fused to GAL4 were treated for 16 h with naringenin. Naringenin enhanced PPARα-LBD activity by 140 ± 5% (p < 0.001). (E) qRT-PCR analysis of JFH1-infected Huh7.5.1 cells treated with 200 µM naringenin for 24 h. Naringenin caused increased expression of PPARα and its target gene ACOX and a concomitant decrease in SREBP and its target HMGR. (F) 1 µM of PPAR antagonist GW9662 inhibited PPRE activity in the presence of 150 µM naringenin by 30% (p < 0.001) and increased ApoB secretion by 78% (p < 0.001). Extracellular HCV infectivity was increased by 34% (p < 0.05) while the accumulation of intracellular infectious HCV particles in the presence of 0.1 µg/ml BFA increased by 50% (p < 0.05).

Fig. 3. Naringenin causes a rapid inhibition of HCV production during long-term treatment

Cells were treated daily for 4 days with 200 µM naringenin or 1000 i.u. IFNα. During the 3 days washout period, all wells were untreated. (A) Viral RNA release into media decreased during treatment, down to 4% with naringenin and 2% with IFNα compared to controls. During washout phase, naringenin-treated samples contained RNA levels similar to controls, suggesting viral replication was unchanged. (B) HCV core levels in media decreased during treatment for naringenin and IFNα. For naringenin-treated cells on days 4, core in the media was reduced to 33 ± 5%. Core accumulation was partly restored during the last day of washout in both naringenin and IFNα-treated samples 87 ± 12%, 52 ± 13%, respectively. (C) ApoB secretion declined during treatment with naringenin. On days 4, ApoB secretion in naringenin-treated cells decreased to 13 ± 1%.

Fig. 4. Naringenin effects on primary human hepatocytes

(A) Naringenin caused a dose-dependent inhibition in ApoB-100 secretion following 24 h of treatment of primary human hepatocytes. Cell viability was >95%. (B) Primary human hepatocytes transfected with PPRE reporter plasmid were treated with naringenin. Naringenin dose-dependently enhanced PPRE activity following 24 h of treatment by 3-fold following 200 and 400 µM naringenin stimulation, not significantly different than 10 µM WY14643 (p = 0.129). (C) Primary human hepatocytes were treated for 7 days with 200 µM naringenin followed by a 2 day washout period. ApoB secretion decreased by 43 ± 8% (p < 0.01) while cell viability remained unchanged (p = 0.137). During the washout period, ApoB secretion returned to 86 ± 15% of basal level (p = 0.174).