M A J O R A R T I C L E
Monoclonal Antibody 2-152a Suppresses
Hepatitis C Virus Infection Through Betaine/
Masaaki Satoh,1Makoto Saito,1Takashi Takano,1,2Yuri Kasama,1Tomohiro Nishimura,1,3Yasumasa Nishito,4
Yuichi Hirata,2Masaaki Arai,2Masayuki Sudoh,5Chieko Kai,6Michinori Kohara,2and Kyoko Tsukiyama-Kohara1
1Department of Experimental Phylaxiology, Faculty of Life Sciences, Kumamoto University, Honjo Kumamoto City;2Department of Microbiology and
Cell Biology, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku;3KAKETSUKEN, Kyokushi, Kikuchi, Kumamoto;4Center for
Microarray Analysis, Tokyo Metropolitan Institute of Medical Science;5Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., Kajiwara,
Kamakura-City, Kanagawa; and6Laboratory of Animal Research Center, Institute of Medical Science, University of Tokyo, Shirokane-dai Minato-Ku,
D24-reductase (DHCR24) by immunizing mice with cells (RzM6-LC) persistently expressing hepatitis C virus
(HCV). Here, we aimed to analyze the activity of 2-152a MAb against HCV replication and explore the molecular
mechanism underlying the antiviral activity.
Methods.We characterized the effects of 2-152a MAb on HCV replication and performed a microarray analysis
of antibody-treated HCV replicon cells. The molecules showing a significant change after the antibody treatment
were screened to examine their relationship with HCV replication.
Results.The antibody had antiviral activity both in vitro and in vivo (chimeric mice). In the microarray
analysis, 2-152a MAb significantly suppressed the expression of betaine/GABA transporter-1 (BGT-1) in 2 HCV
replicon cell lines but not in HCV-cured cells. Silencing of BGT-1 expression by small interfering RNA (siRNA)
revealed significant suppression of HCV replication and infection without cytotoxicity. Further, BGT-1 expression
was significantly increased in the presence of HCV (P , .05).
Conclusions. Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1.
These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV
We recently established a monoclonal antibody (2-152a MAb) that binds to 3b-hydroxysterol-
Hepatitis C virus (HCV) causes chronic hepatitis and
hepatocellular carcinoma (HCC) [1–3]. Chronic HCV
infection isa major globalpublic healthconcern because
it affects at least 170 million people worldwide . The
most effective treatment against HCV currently com-
prises a combination therapy of PEGylated a-interferon
(IFN-a) and ribavirin [4, 5]. However, considering that
sustained virological responses develop in only ap-
proximately half of the patients infected with HCV ge-
notype 1, the clinical efficacy of this therapy is limited
[6, 7]. Efforts to develop therapies against HCV are
further hindered by the high level of viral variation and
capacity of the virus to cause chronic infection.
Therefore, there is an urgent need to develop effective
treatments against chronic HCV infection.
In a previous study, we established a cell line ex-
pressing HCV (RzM6-LC) to investigate the effects of
persistent HCV expression on cell growth . We also
established a monoclonal antibody (2-152a MAb)
against the RzM6-LC cell line to produce clones that
recognize both cell surface and intracellular molecules.
reductase (DHCR24) as the recognition molecule of this
Received 30 November 2010; accepted 10 May 2011.
Correspondence: Kyoko Tsukiyama-Kohara, PhD, DVM, Department of Experi-
mental Phylaxiology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo,
Kumamoto-shi, Kumamoto 860-8556, Japan (firstname.lastname@example.org).
The Journal of Infectious Diseases
? The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
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d Satoh et al
by guest on January 11, 2016
DHCR24 (also termed seladin-1) is an enzyme that catalyzes
the conversion of desmosterol to cholesterol in the postsqualene
cholesterol biosynthetic pathway [9, 10]. DHCR24 also acts as
a hydrogen peroxide scavenger . Therefore, DHCR24 may
play a crucial role in maintaining cell physiology through
cholesterol synthesis and oxidative stress. We previously dem-
onstrated that HCV infection upregulates DHCR24 expression,
and overexpression of DHCR24 inhibits apoptosis and
inactivates the tumor suppressor gene p53 . Moreover,
silencing of DHCR24 suppressed HCV replication . How-
ever, the precise mechanisms through which DHCR24 affects
the HCV life cycle are unclear. In this study, we aimed to analyze
the activity of 2-152a MAb against HCV replication and explore
the molecular mechanism underlying the antiviral activity.
Materials And Methods
Cell Lines and Reagents
Human hepatoma cell line HuH-7 cell-based HCV replicon-
harboring cell lines  R6FLR-N (genotype 1b) , FLR3-1
(genotype 1b) , and JFH-1 (genotype 2a)  were main-
tained in Dulbecco’s modified Eagle’s medium (DMEM)
GlutaMAX (Invitrogen) containing 10% fetal calf serum (FCS;
Sigma-Aldrich) inthe presence ofG418(500mg/mLfor R6FLR-
N and FLR3-1, 300 mg/mL for JFH-1; Invitrogen). Cured/
HuH-7 histone H3 lysine 4 (K4) cells cured off HCV by in-
terferon treatment  were maintained in DMEM GlutaMAX
containing 10% FCS without G418. The JFH/K4 cell line
persistently infected with the HCV JFH-1 strain and HuH-7 cell
lines were maintained in DMEM containing 10% FCS . The
human hepatoblastoma HepG2 cell line was also maintained in
DMEM containing 10% FCS.
Generation of 2-152a MAb
BALB/c strain of mice was immunized with 7–8 intraperitoneal
injections of RzM6-LC cells (5 3 106) in RIBI adjuvant (tre-
halose dimycolate 1 monophosphoryl lipid A emulsion; RIBI
ImmunoChem Research). After completion of the immuniza-
tion regimen, their spleens were excised and splenocytes were
fused with mouse myeloma plasminogen activator inhibitor
(PAI) cells by using PEG1500 (Roche). Hybridoma cells were
then selected with hypoxanthine, aminopterin, and thymidine
(Invitrogen), and culture supernatants were collected for
screening by whole-cell enzyme-linked immunosorbent assay
HCV Infection in Humanized Chimeric Mouse Liver and HCV
mRNA Quantification by Real-time Detection Polymerase Chain
We purchased (from PhoenixBio Co.) chimeric mice that were
established by transplanting human primary hepatocytes into
severely combined immunodeficient (SCID) mice carrying
a urokinase plasminogen activator (uPA) transgene controlled
by an albumin promoter . These mice were then infected
with plasma isolated before 2003 from an HCV-positive patient
(HCR6) [8, 21], in accordance with the Declaration of Helsinki.
The protocols for the animal experiments were preapproved by
the local ethics committee, and the animals were maintained in
accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. HCV genotype 1b RNA
levels were established at 0.96–1.84 3 107copies/mL in mouse
serum samples before the antibody treatment. The antibody
(2-152a MAb) and normal immunoglobin G (IgG, 400 mg/20 g
body weight) were intraperitoneally injected into the mice
(n 5 4) at 2-day intervals over a period of 14 days. IFN-a
(30 mg/kg) was administered subcutaneously at 2-day intervals
over a period of 2 weeks. Human serum albumin in the blood of
chimeric mice was measured by using an Alb-II kit according to
the manufacturer’s instructions (Eiken Chemical). HCV RNA
levels in serum and JFH/K4 cells were measured by real-time
detection polymerase chain reaction (real-time detection
[RTD]-PCR) as described previously . HCV RNA in the cell
cultures and supernatants was extracted by using Isogene and
Isogene LS (Nippon Gene), respectively.
Replication Assay Using HCV Replicon Cells
We used 3 HCV subgenomic replicon cell lines: R6FLR-N,
FLR3-1, and JFH-1. They were seeded at a density of 5 3 103
cells/well in 96-well tissue culture plates in DMEM GlutaMAX
(Invitrogen) containing 5% fetal bovine serum (Thermo Sci-
the medium was removed and serial dilutions of antibody were
added. Luciferase activity was determined by using a Bright-Glo
luciferase assay kit (Promega) after 72 hours according to the
manufacturer’s instructions. The results were calculated as the
average percentage relative to the reactivity in untreated cells,
which was set at 100%. The viability of the replicon cells was
measured by using a WST-8 cell counting kit (Dojindo)
according to the manufacturer’s instructions.
Immunostaining and Antibodies
Cells were cultured on glass coverslips (1.0 cm diameter) and
fixed with 1% paraformaldehyde in phosphate-buffered saline
(PBS) at room temperature for 10 minutes in 24-well plates. To
permeabilize the cell membranes, the cells were treated with 1%
Triton X-100 in PBS at room temperature for 10 minutes. After
washing with 0.05% Tween-20 in PBS, the cells were incubated
with 2-152a MAb, antiprotein disulfate isomerase (PDI) rabbit
polyclonal antibody (Stressgen Bioreagents) or normal mouse
IgG for 1 hour and washed with 0.05% Tween-20 in PBS. Alexa
Fluor 488-labeled goat antimouse IgG was used as the secondary
Anti-NS5Aantibody was providedbyDr YoshiharuMatsuura
(Osaka University). Anti-myc mouse monoclonal antibody
BGT-1 Plays a Role in HCV Replication
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is also thought to be responsible for the hyperosmotic stress
response and in maintaining cell hydration. Denkert et al 
reported that BGT-1 gene expression is induced by hyper-
osmolarity and inhibited by p38 mitogen-activated protein ki-
nase (p38MAPK) inhibitor SB20358. Further, several reports have
evidenced that cell hydration affects viral replication and that
viral replication increases during cell shrinkage due to hyper-
osmolarity, a result that was accompanied by increased BGT-1
mRNA expression . Considering the reduction in HCV
replication by the BGT-1 siRNA treatment, this treatment may
prevent HCV replicationbyaffectinghypoosmotic conditionsin
HCV-infected cells. Further studies are required to examine in
detail the function of BGT-1 in HCV replication.
In summary, we demonstrated that the 2-152a monoclonal
antibody inhibits HCV replication in HCV replicon cells and
mice. The inhibitory effect of the monoclonal antibody on viral
replication may be mediated by the suppression of BGT-1 ex-
pression. We propose BGT-1 as a key target for anti-HCV
Supplementary materialsare availableat TheJournal of Infectious Diseases
online (Supplementary Data).
Supplementary materials consist of data provided by the author that are
published to benefit the reader. The posted materials are not copyedited. The
contents of all supplementary data are the sole responsibility of the authors.
Questions or messages regarding errors should be addressed to the author.
and R. Takehara for their excellent technical support, and Y. Tokunaga for
the insightful comments and helpful discussion.
Financial Support.This work was supported by grants from the
Ministry of Health and Welfare of Japan; Ministry of Education, Culture,
Sports, Science and Technology of Japan; Program for Promotion of
Fundamental Studies in Health Sciences of the National Institute of Bio-
medical Innovation; and Cooperative Research Project on Clinical and
Epidemiological Studies of Emerging and Reemerging Infectious Diseases.
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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