Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit flavonoid naringenin

Abstract

Hepatitis C virus (HCV) infects over 3% of the world population and is the leading cause of chronic liver disease worldwide. HCV has long been known to associate with circulating lipoproteins, and its interactions with the cholesterol and lipid pathways have been recently described. In this work, we demonstrate that HCV is actively secreted by infected cells through a Golgi-dependent mechanism while bound to very low density lipoprotein (vLDL). Silencing apolipoprotein B (ApoB) messenger RNA in infected cells causes a 70% reduction in the secretion of both ApoB-100 and HCV. More importantly, we demonstrate that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, inhibits the microsomal triglyceride transfer protein activity as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and acyl-coenzyme A:cholesterol acyltransferase 2 in infected cells. Stimulation with naringenin reduces HCV secretion in infected cells by 80%. Moreover, we find that naringenin is effective at concentrations that are an order of magnitude below the toxic threshold in primary human hepatocytes and in mice.

Conclusion: These results suggest a novel therapeutic approach for the treatment of HCV infection.

Fig. 1

(A) Immunoprecipitation of Huh7.5.1-secreted ApoB followed by anti-HCV core staining (coimmunoprecipitation). (B) Cell culture secretion of ApoB, HCV-positive strand RNA, and HCV core protein in JFH-1–infected Huh7.5.1 cells in response to oleate, insulin, and brefeldin A. The secretions of ApoB, HCV RNA, and HCV core protein are significantly up-regulated by oleate and down-regulated by insulin in a dose-dependent manner. Brefeldin A, which blocks Golgi-dependent secretion of proteins, significantly inhibits the secretion of ApoB, HCV RNA, and HCV core. Cell viability for all conditions was greater than 90%. (C) Infectivity of cell culture supernatant assessed by colony formation on naïve Huh7.5.1 cells: oleate (0.8 mM), insulin (500 U/L), brefeldin A (2.5 µg/mL), and naringenin (200 µM). **P < 0.01.

Fig. 2

Double immunofluorescence staining of JFH-1–infected Huh7.5.1 cells. (A) Staining for HCV core protein (red). (B) Staining for ApoB100 (green). (C) Superpositioning of the images demonstrates that HCV core protein associates with ApoB100 in the cytoplasm. (D) Relative secretion of ApoB, HCV-positive strand RNA, and HCV core protein in JFH-1–infected Huh7.5.1 cells following silencing of ApoB100 mRNA by SureSilencing shRNA transfection. **P < 0.01.

Fig. 3

(A) Inhibition of ApoB, HCV-positive strand RNA, and HCV core protein secretion by the grapefruit flavonoid naringenin. Naringenin significantly inhibits the secretion of HCV core (P = 0.0001, n = 6) and HCV-positive strand RNA (P = 0.0006, n = 5) in a dose-dependent manner. At the concentration of 200 µM, naringenin inhibited HCV secretion by 80% ± 10%. Cell viability for all conditions was greater than 90%. **P < 0.01. (B) Naringenin inhibits the activity of MTP in a dose-dependent manner. At the concentration of 200 µM, MTP activity was reduced by 58% ± 8% (P = 0.0012, n = 3). (C) Naringenin induces changes in hepatic gene transcription measured by qRT-PCR. HMGR transcription was reduced by 57% ± 3% (P = 0.010, n = 3), whereas the transcription of ACAT2 was reduced by 55% ± 7% (P = 0.016, n = 3). The mRNA levels of actin, MTP, and ACAT1 remained unchanged. Intracellular RNA levels of HCV core also remained unchanged during the 24 hours of treatment. **P < 0.02.

Fig. 4

(A) Naringenin stimulation inhibits ApoB secretion of primary human hepatocytes in a dose-dependent manner. At 200 µM naringenin, ApoB secretion was reduced by 60% ± 7% (P = 0.007, n = 3). (B) Viability of freshly isolated human hepatocytes exposed to increasing concentrations of naringenin for 24 hours. Human hepatocyte viability was 81% ± 3% at 200 µM naringenin and was not judged to be statistically different than the control (77% ± 3%). Human hepatocyte viability dropped significantly only at naringenin concentrations greater than 1000 µM.

Fig. 5

Animal survival and liver enzyme release following intraperitoneal (i.p.) injection of naringenin into 8-week-old male SCID mice. Animals were injected with naringenin at 60, 300, and 1500 mg/kg of body weight. Animals were sacrificed at 48 hours, at which time liver enzymes (AST and ALT) and total triglycerides were analyzed in the animals’ plasma. (A) Animal survival was monitored for several days following injection and was not affected even at the highest dose (1500 mg/kg). The ALT level appeared unchanged over all conditions, whereas AST was found to be slightly elevated at the highest dose. (B) Total triglycerides analyzed in animal plasma 24 hours following injection decreased in response to naringenin.