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

Hepatitis C virus (HCV) infection is a global public health problem, affecting over 3% of the world population. HCV infection develops into a chronic condition in over 70% of the patients, ultimately leading to cirrhosis and hepatocellular carcinoma.1 Current standards of care consist of interferon (α2A) and ribavirin, which have been found to be effective in only 50% of the cases.1 However, this treatment is poorly tolerated by patients and is associated with significant side effects. Therefore, there is a pressing need for the development of alternative strategies for the treatment of HCV infection. HCV has long been known to associate with β-lipoproteins [very low density lipoprotein (vLDL) and low-density lipoprotein (LDL)] circulating in patients’ blood.2 Its E1/E2 receptors have been found to bind to both LDL and high-density lipoprotein,3 whereas HCV core protein has been shown to associate with apolipoprotein AII (ApoAII)4 and lipid droplets in HepG2 cells.5 In addition, HCV replication has been shown to be up-regulated by fatty acids and inhibited by statins; this suggests an interaction between HCV, cholesterol, and lipid metabolism.6 The recent development of an efficient cell culture system in which the full lifecycle of HCV infection is captured has opened new opportunities for the study of the viral secretion.7,8 Using this system, Gastaminza et al.9 demonstrated that intercellular HCV particles have a higher density than their secreted counterparts, suggesting that HCV might bind low-density particles prior to viral egress. Just recently, Huang et al.10 demonstrated that HCV secretion is dependent on both apolipoprotein B (ApoB) expression and vLDL assembly in a chromosomally integrated complementary DNA (cDNA) model of HCV secretion. These results strongly suggest that HCV might be “hitching a ride” along the lipoprotein lifecycle. Therefore, compounds previously shown to influence lipoprotein assembly and secretion could possibly exert a similar effect on HCV. To test this hypothesis, we used the full-length, RNA-based HCV full lifecycle model (JFH1/Huh7.5.1) previously shown to capture important aspects of viral replication, assembly, and infection. Using this model, we demonstrate that HCV is being actively secreted by infected cells in a Golgi-dependent pathway while bound to vLDL. Silencing ApoB messenger RNA (mRNA) by transfection with short hairpin RNA (shRNA) is shown to induce a 70% reduction in the secretion of ApoB, HCV core protein, and HCV RNA. More importantly, we find that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, is able to reduce HCV secretion from infected cells by 80% ± 10%. We demonstrate that naringenin inhibits ApoB secretion by inhibiting the activity of the microsomal triglyceride transfer protein (MTP) as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme reductase (HMGR) and acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2). Moreover, we find that naringenin is effective at a concentration of 200 µM, which is well below its toxic concentration for primary human hepatocytes and severe combined immunodeficient (SCID) mice.