Apolipoprotein B–Dependent Hepatitis C Virus
Secretion Is Inhibited by the Grapefruit Flavonoid
Yaakov Nahmias,1,2Jonathan Goldwasser,1,3Monica Casali,1,2Daan van Poll,1,2Takaji Wakita,4
Raymond T. Chung,2and Martin L. Yarmush1,2
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
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 concen-
trations that are an order of magnitude below the toxic threshold in primary human hepa-
tocytes and in mice. Conclusion: These results suggest a novel therapeutic approach for the
treatment of HCV infection. (HEPATOLOGY 2008;47:1437-1445.)
a chronic condition in over 70% of the patients, ulti-
lic health problem, affecting over 3% of the
Current standards of care consist of interferon (?2A) and
ribavirin, which have been found to be effective in only
50% of the cases.1However, this treatment is poorly tol-
erated by patients and is associated with significant side
effects. Therefore, there is a pressing need for the devel-
HCV has long been known to associate with ?-li-
density lipoprotein (LDL)] circulating in patients’
blood.2Its E1/E2 receptors have been found to bind to
both LDL and high-density lipoprotein,3whereas HCV
tein AII (ApoAII)4and lipid droplets in HepG2 cells.5In
addition, HCV replication has been shown to be up-reg-
an interaction between HCV, cholesterol, and lipid me-
tabolism.6The recent development of an efficient cell
culture system in which the full lifecycle of HCV infec-
tion is captured has opened new opportunities for the
study of the viral secretion.7,8Using this system, Gasta-
minza et al.9demonstrated that intercellular HCV parti-
suggesting that HCV might bind low-density particles
prior to viral egress. Just recently, Huang et al.10demon-
aminotransferase; ApoAII, apolipoprotein AII; ApoB, apolipoprotein B; AST, as-
partate aminotransferase; cDNA, complementary DNA; DMEM, Dulbecco’s mod-
ified Eagle medium; EDTA, ethylene diamine tetraacetic acid; EGF, epidermal
growth factor; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine se-
rum; GFP, green fluorescent protein; HCV, hepatitis C virus; HMGR, 3-hydroxy-
3-methyl-glutaryl-coenzyme reductase; i.p., intraperitoneal; LDL, low-density
lipoprotein; mRNA, messenger RNA; MTP, microsomal triglyceride transfer pro-
tein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; qPCR,
quantitative polymerase chain reaction; SCID, severe combined immunodeficient;
shRNA, short hairpin RNA; Tris, trishydroxymethylaminomethane; vLDL; very
low density lipoprotein.
MA;2Massachusetts General Hospital, Harvard Medical School, Boston, MA;
4National Institute of Infectious Diseases, Tokyo, Japan.
Received May 14, 2007; accepted December 13, 2007.
in Medicine, Massachusetts General Hospital, 114 16th Street, Room 1402,
Charlestown, MA 02129. E-mail: firstname.lastname@example.org; fax: 617-573-9471.
Copyright © 2008 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
Supplementary material for this article can be found on the HEPATOLOGY Web
strated that HCV secretion is dependent on both apoli-
poprotein B (ApoB) expression and vLDL assembly in a
(cDNA) model of HCV secretion.
These results strongly suggest that HCV might be
“hitching a ride” along the lipoprotein lifecycle. There-
fore, compounds previously shown to influence lipopro-
tein 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/
of viral replication, assembly, and infection. Using this
model, we demonstrate that HCV is being actively se-
creted 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 se-
cretion 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 ac-
tivity of the microsomal triglyceride transfer protein
(MTP) as well as the transcription of 3-hydroxy-3-meth-
yl-glutaryl-coenzyme reductase (HMGR) and acyl-coen-
Moreover, we find that naringenin is effective at a con-
centration of 200 ?M, which is well below its toxic con-
centration for primary human hepatocytes and severe
combined immunodeficient (SCID) mice.
Materials and Methods
Reagents and Antibodies. Fetal bovine serum (FBS),
phosphate-buffered saline (PBS), Dulbecco’s modified
Eagle medium (DMEM), penicillin, streptomycin, and
trypsin–ethylene diamine tetraacetic acid (EDTA) were
obtained from Invitrogen Life Technologies (Carlsbad,
CA). Lipoprotein-free FBS was purchased from Biomed-
brefeldin A were purchased from Sigma-Aldrich Chemi-
cals (St. Louis, MO). Immunofluorescence-grade para-
formaldehyde was purchased from Electron Microscope
Sciences (Hatfield, PA). OptiMEM basal medium and
Technologies. The SureSilencing shRNA plasmid kit for
human ApoB [green fluorescent protein (GFP)] was pur-
chased from SuperArray (Frederick, MD). An MTP flu-
orescent activity kit was purchased from Roar Biomedical
icals were purchased from Sigma-Aldrich Chemicals. For
immunoprecipitation, Protein A-Sepharose was pur-
chased from Invitrogen, whereas horseradish peroxidase–
conjugated goat anti-mouse secondary was purchased
from Santa Cruz Biotech (Santa Cruz, CA). For immu-
nofluorescence studies, normal donkey serum and sec-
ondary F(ab?)2 antibody fragments (multiple-labeling
[ML] grade) were obtained from Jackson Immunore-
search (Bar Harbor, ME). Mouse anti-HCV core antigen
(5 ?g/mL) was purchased from US Biological (Swamp-
scott, MA). Goat anti-ApoB (10 ?g/mL) was purchased
from R&D Systems, Inc. (Minneapolis, MN).
Cells and Viruses. The Huh7.5.1 human hepatoma
kindly provided by Dr. Chisari (Scripps Research Insti-
tute, La Jolla, CA) and Dr. Wakita (National Institute of
Huh7.5.1 cells were cultured in DMEM supplemented
streptomycin in a 5% CO2–humidified incubator at
37°C. In vitro transcribed genomic JFH-1 RNA was de-
livered to cells by liposome-mediated transfection as de-
scribed by Zhong et al.8Infected Huh7.5.1 cells were
passaged every 3 days and used at passage ?15. The pres-
ence of HCV in these cells and corresponding superna-
immunofluorescence staining. Primary human hepato-
culture medium composed of DMEM supplemented
with 10% heat-inactivated FBS, 200 U/mL penicillin/
streptomycin, 7.5 ?g/mL hydrocortisone, 20 ng/mL epi-
dermal growth factor (EGF), 14 ng/mL glucagons, and
0.5 U/mL insulin. The medium was supplemented with
2% dimethyl sulfoxide for long-term culture of the pri-
HCV Secretion. HCV-infected Huh7.5.1 cells were
plated on a 6-well plate at a density of 1?105cells/cm2
and cultured overnight in the standard medium. Prior to
the beginning of the experiment, the cells were washed 3
times with PBS and cultured with DMEM containing
5% lipoprotein-free FBS. Oleate, insulin, naringenin,
text. Following 24 hours of incubation, the plate was
gently agitated to release mechanically bound particles,
and the medium was collected, filtered to remove cellular
debris, and stored at ?80°C for further analysis. The
attached cells were washed 3 times with PBS, harvested,
pelleted, and stored at ?80°C for further analysis.
Coimmunoprecipitation. The binding of Huh7.5.1-
secreted JFH1 particles to ApoB was assessed with coim-
1438NAHMIAS ET AL.HEPATOLOGY, May 2008
munoprecipitation. Anti-human ApoB-100 antibody (5
?g) was bound to 100 ?L of Protein A-Sepharose on ice.
Three milliliters of the JFH1-infected Huh7.5.1 condi-
tioned medium (1?106cells/mL) was added to the mix-
ture, which was subsequently rotated for 4 hours at 4°C.
The sample was spun down at 10,000g in a microcentri-
fuge and washed 3 times with 50 mM trishydroxymeth-
ylaminomethane (Tris)-HCl (pH 7.5) containing 5 mM
EDTA. Finally, the sample was eluted in 100 ?L of 10
mM Tris-HCl (pH 8.5) containing sodium dodecyl sul-
fate. The protein concentration in the eluted buffer was
quantified as described later, and 20 ?g of protein was
loaded onto a 7.5% Tris-HCL resolving gel. Resolved
proteins were transferred to a polyvinylidene fluoride
membrane and stained against HCV core (0.5 ?g/mL).
particles was measured as previously described.8Naı ¨ve
Huh7.5.1 cells were grown to 80% confluence and ex-
posed to cell culture supernatants diluted 10-fold in the
the medium was replaced, and the cells were cultured for
3 additional days. Levels of HCV infection were deter-
mined by immunofluorescence staining for HCV core
protein. The viral titer is expressed as focus forming units
per milliliter of supernatant.
Human ApoB Enzyme-Linked Immunosorbent As-
say (ELISA). Huh7.5.1–secreted and primary human
hepatocyte–secreted ApoB was detected in the medium
with the ALerCHEK, Inc. (Portland, ME), total human
ApoB ELISA kit. The medium was diluted 1:10 with the
specimen diluent, and the assay was carried out according
to the manufacturer’s directions.
HCV core antigen was detected in the medium with the
Wako Chemicals (Cambridge, MA) ORTHO HCV an-
was carried out according to the manufacturer’s direc-
Total Protein Assay. The total protein content of the
cells was measured with the Bio-Rad Laboratories (Her-
cules, CA) protein assay based on the Bardford method.
Briefly, a cell pellet was lysed in 350 ?L of 0.1% Triton
X-100, and 5-?L samples were loaded onto a 96-well
plate and incubated for 15 minutes with 250 ?L of Coo-
measured at 595 nm and compared to a bovine serum
Quantitative, Real-Time, Reverse-Transcription
Polymerase Chain Reaction (PCR). Virus samples col-
lected in each experiment were filtered with a 0.45-?m
at 95°C for 45 minutes. The reverse-transcription reac-
tion step was performed on a Mastercycler epgradientS
(Eppendorf) instrument using Omniscript and Sensis-
a Light Cycler LC-24 (Idaho Technology) using Super-
Script III Platinum CellsDirect Two-Step qRT-PCR kits
(Invitrogen). For the reverse-transcription step, 2 ?L of a
sample without RNA extraction was used. For real-time
PCR, 1 ?L of the reverse-transcription reactions was
used. All reactions were performed according to the
manufacturer’s instructions with the primers detailed
in Table 1.
Cellular Viability. The viability of both Huh7.5.1
cells and primary human hepatocytes was studied with
Thermo Fisher Scientific (Waltham, MA) Infinity aspar-
tate aminotransferase (AST) liquid reagent. Medium
samples (15 ?L/well) were loaded onto a 96-well plate in
triplicates and mixed with 150 ?L of the AST liquid
reagent. Absorbance decay was measured at the wave-
length of 340 nm with 15-second intervals in a Bio-Rad
Benchmark Plus spectrophotometer. Values were nor-
malized to the total amount of AST available per culture,
which was determined by total cell lysis induced by 1%
Triton X-100 for 20 minutes at room temperature. Cell
viability for all conditions reported in the Results section
was greater than 90%.
MTP Activity Assay. MTP activity was analyzed with
an MTP assay kit as previously described.11The assay is
acceptor particles due to MTP activity. Briefly, confluent
Huh7.5.1 cells were stimulated with naringenin or a carrier
and scraped off the dish with a cell scraper. Samples were
taining protease inhibitors. The MTP assay was performed
donor and acceptor solutions in 250 ?L of total buffer (15
mM Tris, pH 7.4; 40 mM NaCl; 1 mM EDTA). The in-
Table 1. PCR Primers
HEPATOLOGY, Vol. 47, No. 5, 2008NAHMIAS ET AL.1439
37°C at the excitation wavelength of 465 nm and emission
wavelength of 538 nm.
Animal Studies. Male SCID mice (8 weeks old,
20-25 g) were obtained from Charles River Laboratories
(Wilmington, MA). Animals were treated in accordance
with National Institutes of Health guidelines and the
Massachusetts General Hospital Subcommittee on Re-
search Animal Care. The mice were allowed free access to
laboratory chow and water ad labium. Naringenin was
intraperitoneal injection. Two days following the treat-
ment, animals were sacrificed, and blood was withdrawn
by cardiac puncture. AST and alanine aminotransferase
(ALT) enzyme levels were assessed as described previ-
ously. Total triglycerides were measured with a kit pur-
chased from Sigma-Aldrich Chemicals according to the
Silencing ApoB mRNA. HCV-infected Huh7.5.1
cells were plated in T-25 tissue culture flasks at a density
of 1?105cells/cm2and cultured overnight in the stan-
dard medium. Prior to silencing, the cells were washed 3
times with PBS, and the medium was replaced with Op-
mids against human ApoB100 as well as shRNA plasmid
control (500 ng/mL) were combined with Lipofectamine
2000 in OptiMEM and incubated with the cells over-
night. SureSilencing shRNA plasmids code for GFP,
Research Center. Transfected cells (10% of the total pop-
ulation) were sorted directly into a 12-well plate and al-
lowed to adhere overnight. The culture medium was
conditioned by the transfected cells for 24 hours and an-
alyzed as described previously.
Immunofluorescence Microscopy. Huh7.5.1 cells
were washed 3 times with PBS and fixed in 4% electron
microscopy–grade paraformaldehyde for 10 minutes at
incubated in 100 mmol/L glycine for 15 minutes to sat-
urate reactive groups. Samples were permeabilized for 15
with 1% bovine serum albumin and 5% donkey serum at
room temperature, and stained with primary antibodies
overnight at 4°C. After additional washes with PBS, sam-
ples were stained with fluorescently tagged secondary an-
tibodies for 45 minutes at room temperature.
Huh7.5.1-Secreted HCV Is Bound to ApoB. Recent
ApoB expression and vLDL assembly to occur.10There-
fore, HCV secreted by the JFH1/Huh7.5.1 full viral life-
cycle model could potentially be secreted while bound to
vLDL. To determine if Huh7.5.1-produced HCV is
bound to vLDL, we immunoprecipitated the Huh7.5.1-
conditioned medium against human ApoB antibodies
and detected bound HCV core protein in the eluted sam-
ple. The results presented in Fig. 1A demonstrate that
HCV core protein is bound to ApoB-100 in our samples.
HCV core could not be detected when the sample was
precipitated against irrelevant antibody (control) but was
easily detected in the cell medium (JFH1).
HCV Secretion Mirrors That of vLDL. The inter-
action between HCV and ApoB suggests that the virus
might be actively secreted by the cells while bound to
vLDL. However, the interaction between these particles
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 brefel-
din 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
naı ¨ve Huh7.5.1 cells: oleate (0.8 mM), insulin (500 U/L), brefeldin A
(2.5 ?g/mL), and naringenin (200 ?M). **P ? 0.01.
1440NAHMIAS ET AL.HEPATOLOGY, May 2008
might also occur outside the cell. To determine if HCV is
being actively secreted by the cells while bound to vLDL,
stimulation, which was previously shown to oppositely
modulate ApoB secretion in culture.12Figure 1B shows
ApoB, HCV core, and HCV-positive strand RNA secre-
tion by Huh7.5.1 cells infected with the JFH-1 virus. As
expected, ApoB secretion is significantly up-regulated by
oleate (P ? 0.0023, n ? 5) and down-regulated by insu-
lin (P ? 0.0073, n ? 5) in a dose-dependent manner.
Similarly, HCV core protein secretion is significantly up-
regulated by oleate (P ? 0.0073, n ? 3) and down-regu-
lated by insulin (P ? 0.0223, n? 3) in a dose-dependent
manner. The secretion of HCV-positive strand RNA,
intracellular levels of HCV RNA remained unchanged
following both treatments.
Brefeldin A is a commonly used toxin that disrupts
communication between the endoplasmic reticulum and
the Golgi, inhibiting the active secretion of proteins.12,13
blocked ApoB secretion (P ? 0.0001, n ? 5). Interest-
ingly, brefeldin A significantly inhibits the secretion of
HCV core protein (P ? 0.0021, n ? 4) and HCV-posi-
tive strand RNA (P ? 0.0006, n ? 3). To assess whether
the changes in HCV core protein and RNA secretion
natant, we measured the ability of the secreted virus to
infect naı ¨ve Huh7.5.1 cells. Figure 1C shows that the
infectivity of the cell supernatant increased following
oleate stimulation, decreased because of insulin, and was
strongly inhibited following brefeldin A stimulation by
89% ? 10% (P ? 0.001, n ? 3). These results suggest
that HCV is being actively secreted by the cells, perhaps
while bound to vLDL.
HCV Core Antigen Colocalizes with ApoB. Previ-
ously, HCV core protein was shown to associate with
ApoAII4and lipid droplets in HepG2 cells5overexpress-
ing the core protein. Just recently, Huang et al.10demon-
strated that HCV core protein colocalizes with ApoB in a
chromosomally integrated cDNA model of HCV. To as-
certain if HCV core protein associates with ApoB in
JFH-1 virus–infected Huh7.5.1 cells, we double-stained
Huh7.5.1 cells 2 days post infection by immunofluores-
cence for both viral and native proteins. Figure 2 demon-
strates the colocalization of HCV’s core and ApoB100 in
infected cells. HCV core protein associates with areas in
note that although the proteins appear to be closely asso-
ciated, we fail to find a one-to-one correspondence be-
tein as well as previous data suggests that HCV might be
“tagging along” ApoB secretion. Therefore, silencing
ApoB production in the cell might decrease HCV secre-
tion. Figure 2D demonstrates a 69% ? 6% decrease in
shRNA (P ? 0.0001, n ? 3). Interestingly, HCV core
protein secretion was significantly decreased by 75% ?
4% at the same time (P ? 0.0002, n ? 3). HCV-positive
strand RNA secretion was also significantly decreased by
69% ? 4% (P ? 0.0015, n ? 3).
HCV Secretion Is Inhibited by Naringenin. Narin-
cholesterol levels both in vivo14and in vitro.15It is
thought that naringenin inhibits ApoB secretion by re-
ducing the activity and expression of MTP and
ACAT.15,16To assess if naringenin inhibits HCV secre-
tion in a similar manner, we cultured infected Huh7.5.1
demonstrates that naringenin inhibits the secretion of
RNA (P ? 0.0006, n ? 5) in a dose-dependent manner.
At the concentration of 200 ?M, naringenin inhibited
HCV secretion by 80% ? 10%. Interestingly, intracellu-
intracellular HCV core protein expression (Supplemen-
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.
HEPATOLOGY, Vol. 47, No. 5, 2008NAHMIAS ET AL.1441
tary Fig. 1) remained unchanged. To assess whether the
naringenin-induced inhibition of HCV core protein and
RNA secretion correlated with changes of viral infectivity
in the cell supernatant, we measured the ability of the
secreted virus to infect naı ¨ve Huh7.5.1 cells. Figure 1C
shows that the infectivity of the cell supernatant was
strongly inhibited following naringenin stimulation by
79% ? 10% (P ? 0.0018, n ? 3).
Although the activity of naringenin has been described
in uninfected cells,15,17,18it has yet to be characterized in
HCV-infected cells. Figure 3B demonstrates that narin-
At the concentration of 200 ?M, MTP activity was re-
duced by 58% ? 8% (P ? 0.0012, n ? 3). In addition,
we demonstrate that naringenin induces significant
changes in hepatic gene transcription measured by qRT-
PCR (Fig. 3C). HMGR transcription was reduced by
57% ? 3% (P ? 0.010, n ? 3), whereas ACAT2 was
reduced by 55% ? 7% (P ? 0.016, n ? 3). In contrast,
the mRNA levels of actin, MTP, ACAT1, and HCV re-
Naringenin Does Not Display Hepatic or In Vivo
Toxicity. To assess the potential of naringenin-based
treatment, we measured ApoB secretion in primary hu-
man hepatocytes following 24 hours of stimulation with
naringenin. Figure 4A demonstrates a dose-dependent
decrease in ApoB secretion following naringenin stimula-
by 60% ? 7% (P ? 0.007, n ? 3). The viability of
primary human hepatocytes exposed to increasing con-
centrations of naringenin is shown in Fig. 4B. Human
hepatocyte viability was 81% ? 3% at 200 ?M naringe-
nin and was not judged to be statistically different than
that of the control (78% ? 3%). Human hepatocyte vi-
ability dropped significantly only at naringenin concen-
trations greater than 1000 ?M.
To further assess naringenin potential, we delivered
naringenin by intraperitoneal injection to 8-week-old
male SCID mice at concentrations of 60, 300, and
1500 mg/kg (approximately 200, 1000, and 5000
?M). Animal survival was not affected by naringenin at
these doses. To discern if liver damage occurred, we
measured levels of AST and ALT in the animals’
plasma 48 hours following injection. Figure 5 demon-
strates that there was no elevation of ALT levels under
all conditions. AST levels appeared to increase but re-
mained under 100 U/L even at the highest dose. To
assess naringenin’s ability to reduce circulating vLDL
levels, we measured total triglyceride levels in animal
plasma. Figure 5A demonstrates a decrease in triglyc-
erides following naringenin injection.
Fig. 3. (A) Inhibition of ApoB, HCV-positive strand RNA, and HCV core
protein secretion by the grapefruit flavonoid naringenin. Naringenin sig-
nificantly 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 ?M, 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 ?M, 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 un-
changed during the 24 hours of treatment. **P ? 0.02.
1442 NAHMIAS ET AL. HEPATOLOGY, May 2008
HCV is a leading cause of chronic liver disease world-
wide. Although the disease develops to cirrhosis in only
20% of the cases, the sheer scope of infection and lack of
A simulation of the US population for the years 2010-
2019 predicts nearly 200,000 deaths associated with
HCV infection and direct medical expenditures in excess
need for the development of alternative strategies for the
treatment of HCV infection.
The interaction between HCV infection, cholesterol,
and fatty acid metabolism has received significant atten-
tion, mainly because of the development of liver steatosis
in chronically infected patients.1However, the lack of an
efficient cell culture model of HCV replication and infec-
tion has significantly limited research in the field. Despite
these limitations, several groups have demonstrated that
HCV core protein associates with ApoAII4and lipid
droplets in HepG2 cells5overexpressing the protein. The
data suggest that HCV in infected patients might circu-
late as lipoviral particles.19The development of HCV
replicon systems20has allowed for the efficient study of
viral replication in culture. Using this system, Kapadia
and Chisari6demonstrated that HCV replication is regu-
lated by geranylgeranylation and fatty acid metabolism.
Others have demonstrated that HCV nonstructural pro-
teins, such as nonstructural protein 5A, inhibit ApoB se-
The recent development of the JFH-1 virus7in com-
bination with the Huh7.5.1 cell line8has allowed for the
titers in culture. This model allows for the identification
of intercellular infectious HCV particles with a higher
density than that of their secreted counterparts,9suggest-
Fig. 4. (A) Naringenin stimulation inhibits ApoB secretion of primary human hepatocytes in a dose-dependent manner. At 200 ?M 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 ?M 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 ?M.
Fig. 5. Animal survival and liver enzyme release following intraperito-
neal (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.
HEPATOLOGY, Vol. 47, No. 5, 2008NAHMIAS ET AL. 1443
ing the binding of HCV to low-density particles in the
onstrated that HCV assembled in ApoB and MTP en-
riched vesicles and that the viral secretion was dependent
on both ApoB expression and vLDL assembly in a chro-
the association between HCV and serum ?-lipoproteins
(vLDL and LDL) is well known,2these results strongly
cholesterol lifecycle. This hypothesis is intriguing as it
might explain the presence of HCV in intestinal cells, a
second site of lipoprotein production.22In addition, it
ger receptor class B type I,25and heparin sulfate.26
Our results strongly support this hypothesis. We dem-
onstrate that HCV produced by the Huh7.5.1 cell line is
bound to ApoB and that its secretion is inhibited by
brefeldin A, a metabolite of the fungus Eupenicillium
brefeldianum, which blocks the communication between
the endoplasmic reticulum and the Golgi, effectively in-
hibiting protein secretion.12,13We also demonstrate that
down-regulated by insulin, precisely mirroring ApoB se-
cretion by the cells.12Moreover, silencing ApoB100
mRNA caused a significant and parallel decrease in HCV
tion pathway suggests a novel therapeutic approach for
the treatment of HCV infection.
Naringin, one of the most abundant flavonoids in cit-
rus fruits, is hydrolyzed by enterobacteria to naringenin
prior to being absorbed. Naringenin has been reported to
be an antioxidant,27MTP and ACAT inhibitor,16and
regulator of cytochrome P4503A and 4A activity.28,29
The ability of naringenin, or its glycosylated form, to
significantly reduce plasma cholesterol levels has been
demonstrated both in vivo and in vitro.14,15It is thought
that naringenin inhibits the expression and activity of
MTP, which catalyzes the transfer of lipids to the nascent
ApoB molecule as it buds into the endoplasmic reticulum
as a vLDL particle.16-18Our results demonstrate that
short-term (24-hour) stimulation of infected hepatocytes
with 200 ?M naringenin significantly inhibits HCV se-
cretion by 80% ? 10% and the infectivity of the titer by
79% ? 10%. At the same time, transcription of the viral
RNA remains unchanged. We suggest that this is due in
part to the inhibition of MTP activity by 58% ? 8% as
well as the inhibition of HMGR and ACAT2 transcrip-
tion. To further demonstrate naringenin as a potential
therapy, we show that the compound is nontoxic to
freshly isolated human hepatocytes up to concentrations
greater than 1000 ?M. In addition, we demonstrate that
tion by primary human hepatocytes.
The concept of supplementing HCV patients’ diets
with naringenin is appealing. A recent clinical trial in
hypercholesterolemic patients demonstrated that a low
dose of naringin (400 mg/day) lowered LDL levels by
17%.30A similar cholesterol-lowering effect of naringe-
nin was demonstrated in rabbits14,31and rats.32However,
it is worth noting that the absorbance of naringenin
through the intestinal wall is limited (less than 8%), and
this suggests that short-term therapeutic doses would
gested that the median lethal dose (50% kill) for naringe-
nin is 2000 mg/kg for both rats and guinea pigs by
intraperitoneal injection.33Our results show that doses
up to 1500 mg/kg naringenin given by intraperitoneal
injection to mice did not cause death or a marked eleva-
tion of liver enzymes, suggesting that intravenous admin-
istration of naringenin is in the realm of possibility.
The ability of the liver to regenerate in the context of
the RNA-based lifecycle of HCV allows for the potential
occurs in about 30% of HCV-infected patients. The pos-
sible reduction of HCV viral load by inhibiting viral se-
cretion could allow uninfected cells to regenerate,
potentially increasing the overall rate of viral clearance.
Future studies would focus on the long-term ability of
naringenin and perhaps other citrus flavonoids to reduce
viral load in animal models, such as the KMT Mouse
model,34and long-term cultures of primary human hepa-
his help with both fluorescence-activated cell sorting and
western blotting. We also thank Dr. Francis Chisari
(Scripps Research Institute) for the Huh7.5.1 cell line.
Microscopic imaging studies were made possible by the
Core Morphology Facility of the Boston Shriners Hospi-
We thank Chris Pohun Chen for
1. Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hep-
atitis. Annu Rev Pathol Mech Dis 2006;1:23-61.
2. Thomssen R, Bonk S, Propfe C, Heermann KH, Kochel HG, Uy A.
Association of hepatitis C virus in human sera with beta-lipoprotein. Med
Microbiol Immunol 1992;181:293-300.
al. Binding of human lipoproteins (low, very low, high density lipopro-
teins) to recombinant envelope proteins of hepatitis C virus. Med Micro-
biol Immunol 2000;188:177-184.
4. Sabile A, Perlemuter G, Bono F, Kohara K, Demaugre F, Kohara M, et al.
is modulated by fibrates. HEPATOLOGY 1999;30:1064-1076.
5. Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura Y, et al.
Hepatitis C virus core protein shows a cytoplasmic localization and asso-
1444NAHMIAS ET AL.HEPATOLOGY, May 2008
ciates to cellular lipid storage droplets. Proc Natl Acad Sci U S A 1997;94: Download full-text
host geranylgeranylation and fatty acids. Proc Natl Acad Sci U S A 2005;
7. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, et al.
Production of infectious hepatitis C virus in tissue culture from a cloned
viral genome. Nat Med 2005;11:791-796.
8. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, et al.
Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A
of infectious intracellular and secreted hepatitis C virus particles. J Virol
10. Huang H, Sun F, Owen DM, Li W, Chen Y, Gale M Jr, et al. Hepatitis C
virus production by human hepatocytes dependent on assembly and secre-
tion of very low-density lipoproteins. Proc Natl Acad Sci U S A 2007;104:
11. Perlemuter G, Sabile A, Letteron P, Vona G, Topilco A, Chretien Y, et al.
Hepatitis C virus core protein inhibits microsomal triglyceride transfer
protein activity and very low density lipoprotein secretion: a model of
viral-related steatosis. FASEB J 2002;16:185-194.
B-containing lipoproteins: information obtained from cultured liver cells.
J Lipid Res 1993;34:167-179.
13. Misumi Y, Misumi Y, Miki K, Takatsuki A, Tamura G, Ikehara Y. Novel
blockade by brefeldin A of intracellular transport of secretory proteins in
cultured rat hepatocytes. J Biol Chem 1986;261:11398-11403.
14. Kurowska E, Borradaile N, Spence JD, Carroll KK. Hypocholesterolemic
effects of dietary citrus juices in rabbits. Nutr Res 2000;20:121-129.
15. Allister EM, Borradaile NM, Edwards JY, Huff MW. Inhibition of micro-
somal triglyceride transfer protein expression and apolipoprotein B100
secretion by the citrus flavonoid naringenin and by insulin involves activa-
tion of the mitogen-activated protein kinase pathway in hepatocytes. Di-
activity and expression of ACAT2 and MTP. J Lipid Res 2001;42:725-
17. Borradaile NM, Dreu LED, Barrett PHR, Huff MW. Inhibition of hepa-
tocyte apoB secretion by naringenin: enhanced rapid intracellular degra-
dation independent of reduced microsomal cholesteryl esters. J Lipid Res
18. Borradaile NM, Dreu LED, Barrett PHR, Behrsin CD, Huff MW. Hepa-
flavonoid, naringenin, via inhibition of MTP-mediated microsomal tri-
glyceride accumulation. Biochemistry 2003;42:1283-1291.
virus particles and lipoprotein metabolism. Semin Liver Dis 2005;25:93-
Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
proteins inhibit apolipoprotein B100 secretion. J Biol Chem 2005;280:
22. Deforges S, Evlashev A, Perret M, Sodoyer M, Pouzol S, Scoazec JY, et al.
Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo.
J Gen Virol 2004;85(pt 9):2515-2523.
23. Nahmias Y, Casali M, Barbe L, Berthiaume F, Yarmush ML. Liver endo-
thelial cells promote LDL-R expression and the uptake of HCV-like par-
ticles in primary rat and human hepatocytes. HEPATOLOGY 2006;43:257-
24. Agnello V, Abel G, Elfahal M, Knight GB, Zhang QX. Hepatitis C virus
and other flaviviridae viruses enter cells via low density lipoprotein recep-
tor. Proc Natl Acad Sci U S A 1999;96:12766-12771.
25. Maillard P, Huby T, Andre ´o U, Moreau M, Chapman J, Budkowska A.
SR-BI/Cla1 is mediated by ApoB containing lipoproteins. FASEB J 2006;
26. Barth H, Schnober EK, Zhang F, Linhardt RJ, Depla E, Boson B, et al.
Viral and cellular determinants of the hepatitis C virus envelope-heparan
sulfate interaction. J Virol 2006;80:10579-10590.
27. Kanno S-I, Tomizawa A, Hiura T, Osanai Y, Shouji A, Ujibe M, et al.
Inhibitory effects of naringenin on tumor growth in human cancer cell
lines and sarcoma S-180-implanted mice. Biol Pharm Bull 2005;28:527-
28. Moon YJ, Wang X, Morris ME. Dietary flavonoids: effects on xenobiotic
and carcinogen metabolism. Toxicol In Vitro 2006;20:187-210.
29. Huong DT, Takahashi Y, Ide T. Activity and mRNA levels of enzymes
involved in hepatic fatty acid oxidation in mice fed citrus flavonoids. Nu-
30. Jung UJ, Kim HJ, Lee JS, Lee MK, Kim HO, Park EJ, et al. Naringin
supplementation lowers plasma lipids and enhances erythrocyte antioxi-
dant enzyme activities in hypercholesterolemic subjects. Clin Nutr 2003;
31. Lee C-H, Jeong T-S, Choi Y-K, Hyun B-H, Oh G-T, Kim E-H, et al.
Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, as-
sociated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high
cholesterol-fed rabbits. Biochem Biophys Res Commun 2001;284:681-
32. Kim S-Y, Kim H-J, Lee M-K, Jeon S-M, Do G-M, Kwon E-Y, et al.
Naringin time-dependently lowers hepatic cholesterol biosynthesis and
plasma cholesterol in rats fed high-fat and high-cholesterol diet. J Med
33. EKMMA8 Eksperimentalna Meditsina i Morfologiya. Vol 19. Sofia, Bul-
garia: Hemus; 1980:207.
34. Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, et al.
HEPATOLOGY, Vol. 47, No. 5, 2008 NAHMIAS ET AL.1445