Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies
Unlabelled: Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection where transmission is potentiated by cell-cell contact. To define route(s) of transmission, HCV was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by >95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess whether cell-cell transfer of viral infectivity occurs, HCV-infected cells were cocultured with fluorescently labeled naïve cells in the presence or absence of nAbs. Enumeration by flow cytometry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immunoglobulins from HCV(+) patients. The host cell molecule CD81 and the tight junction protein Claudin 1 (CLDN1) are critical factors defining HCV entry. Soluble CD81 and anti-CD81 abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection. CD81-negative HepG2 hepatoma cells were resistant to cell-free virus infection but became infected after coculturing with JFH-infected cells in the presence of nAb, confirming that CD81-independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-CLDN1 targets suggested that cell-cell transmission is dependent on CLDN1 expression. Conclusion: These data suggest that HCV can transmit in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route for evading nAbs.
Hepatitis C Virus Cell-Cell Transmission in Hepatoma
Cells in the Presence of Neutralizing Antibodies
Jennifer M. Timpe,
Michelle J. Farquhar,
Helen J. Harris,
Geert Leroux Roels,
and Jane A. McKeating
Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection
where transmission is potentiated by cell-cell contact. To deﬁne route(s) of transmission, HCV
was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize
cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by
>95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess
whether cell-cell transfer of viral infectivity occurs, HCV-infected cells were cocultured with
ﬂuorescently labeled naı¨ve cells in the presence or absence of nAbs. Enumeration by ﬂow cytom-
etry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immu-
noglobulins from HCV
patients. The host cell molecule CD81 and the tight junction protein
Claudin 1 (CLDN1) are critical factors deﬁning HCV entry. Soluble CD81 and anti-CD81
abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection.
CD81-negative HepG2 hepatoma cells were resistant to cell-free virus infection but became
infected after coculturing with JFH-infected cells in the presence of nAb, conﬁrming that CD81-
independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-
CLDN1 targets suggested that cell-cell transmission is dependent on CLDN1 expression.
Conclusion: These data suggest that HCV can transmit in vitro by at least two routes, cell-free
virus infection and direct transfer between cells, with the latter offering a novel route for evading
epatitis C virus (HCV) has emerged as the ma-
jor etiological agent of liver disease. Approxi-
mately 170 million individuals are infected
worldwide, and the majority are at risk for developing
serious progressive liver disease, with HCV being the
leading indication for liver transplantation. The HCV
single-stranded RNA genome encodes a single polypro-
tein, which is cleaved by viral and cellular proteases to
produce the structural proteins; core E1 and E2 and non-
structural proteins; p7, NS2, NS3, NS4A, NS4B, NS5A,
and NS5B. The only approved treatment for HCV infec-
tion is interferon-
in combination with ribavirin, which
is toxic and only effective in 50% of individuals with
genotype I infections. Clearly, there is a need for more
effective therapies and for the development of prophylac-
tic and/or therapeutic vaccines.
Cellular and humoral responses are generated during
acute infection, but they are insufﬁcient to achieve viral
clearance in the majority of individuals, with approxi-
mately 60%-80% of new infections becoming persis-
Neutralizing antibody (nAb) responses often
provide the ﬁrst-line adaptive defense against infection by
limiting virus spread. However, little is known about the
impact of the humoral immune response on HCV patho-
biology. Serum antibodies (Abs) from chronically HCV-
infected individuals demonstrate broadly reactive
neutralizing properties in vitro and yet fail to control viral
infection in vivo.
The reasons for their lack of effect are
poorly understood. HCV may escape neutralization by
Abbreviations: Ab, antibody; CFSE, carboxyﬂuorescein diacetate succinimidyl ester;
CLDN1, Claudin 1; CMFDA, 5-chloromethylﬂuorescein diacetate; DEN3, dengue
virus type-3; DMEM, Dulbecco’s modiﬁed Eagle’s medium; FBS, fetal bovine serum;
HCV, hepatitis C virus;HCVcc, cell culture–grownhepatitisC virus; HCVpp,hepatitis
C virus pseudotype; HTLV-1, human T cell leukemia virus type I; IU, infectious unit;
mAb, monoclonal antibody; MFI, mean ﬂuorescence intensity; MLVpp, murine leuke-
mia virus pseudoparticle; nAb, neutralizing antibody; pi., post infection;sCD81, soluble
CD81; SEM, standard error of the mean; SI, speciﬁc infectivity.
Institute for Biomedical Research, University of Birmingham, Bir
mingham, United Kingdom; and
Center for Vaccinology, Ghent University and
Hospital, Ghent, Belgium.
Received April 16, 2007; accepted August 1, 2007.
Supported by the Medical Research Council (United Kingdom) and the Wellcome
Address reprint requests to: Jane A. McKeating, Ph.D., Institute for Biomedical
Research, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
E-mail: firstname.lastname@example.org; fax: (44) 121 414 3599.
Copyright © 2007 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 H
conventional genetic mutation
or other less direct eva-
Viruses can disseminate within a host by two mecha-
nisms: release of cell-free virions or direct passage between
infected and uninfected cells. In general, direct cell-cell
transfer is considered more rapid and efﬁcient than cell-
free spread because it obviates rate-limiting early steps in
the virus life cycle, such as virion attachment.
cell-cell transfer of viral infectivity may allow viruses to
evade elements of the immune response, such as nAbs and
complement. For viruses such as human T cell leukemia
virus type I (HTLV-I), cell-cell infection appears to be the
principal mode of dissemination both within and be-
Recent developments have allowed HCV to be propa-
gated in cell cultures [cell culture–grown hepatitis C virus
allowing studies on viral transmission.
HCV infection of hepatoma cells results in focal areas of
infection that are potentiated by cell-cell contact, and this
suggests localized transmission between adjacent cells.
The ability of HCV to transmit to naı¨ve target cells when
cultured in the presence of an agarose overlay or nAbs
suggests that direct cell-cell routes of HCV transmission
exist. The host molecule CD81 and the tight junction
protein Claudin 1 (CLDN1) are reported to be critical
factors deﬁning HCV entry.
Experiments to address
the receptor dependency of cell-cell transmission suggest
that CLDN1 is required but that CD81-dependent and
independent routes exist. These data support cell-free and
cell-cell routes of HCV transmission in vitro and raise
questions on the mechanism of HCV spread in vivo and
the effectiveness of prophylactic Abs and agents targeting
the entry step of the life cycle.
Materials and Methods
Cells and Reagents. HeLa, HepG2, and 293T cells
were obtained from the American Type Culture Collec-
tion and propagated in 10% fetal bovine serum (FBS)/
Dulbecco’s modiﬁed Eagle’s medium (DMEM). Huh-
7.5 cells were provided by Dr. Rice (Rockefeller
and propagated in 10% FBS/DMEM/1%
nonessential amino acids. Abs and recombinant proteins
were provided as follows: anti-NS5A 9E10
Rockefeller University), anti-core JM122 (Dr.
McLauchlan, Medical Research Council (MRC) Institute
for Virology), anti-E2 C1 and dengue virus type-3
(DEN3) monoclonal antibodies (mAbs; Dr. Burton,
Scripps Research Institute),
anti-CD81 M38 (Dr. Ber-
ditchevski, Birmingham University), and anti-CLDN1
JAY.8 (Zymed) and soluble CD81 (sCD81; Dr. Liu,
Massachusetts Institute of Technology).
lin was puriﬁed from two HCV-infected patients (P68
and P70, genotype 1a) and a noninfected individual
(NC2) by protein A chromatography (Amersham).
HCV Genesis and Infection. Retroviral pseudotypes
bearing HCV glycoproteins [hepatitis C virus pseudotype
(HCVpp)] were generated by transfection of plasmids en-
coding human immunodeﬁciency virus provirus express-
ing luciferase and E1E2 glycoproteins as previously
HCVcc strains J6/JFH
erated by electroporation of transcribed RNA from full-
length genomes (Megascript-T7 kit, Ambion) into Huh-
7.5 cells as previously described.
72-96 hours post electroporation were stored at !80°C.
Huh-7.5 cells were seeded at 0.75 " 10
1.5 " 10
(standard density), or 3 " 10
. After 24 hours, cells were infected for 1 hour
with J6/JFH or JFH diluted in 3% FBS/DMEM at a
multiplicity of infection of 0.01. Unbound virus was re-
moved, and fresh 3% FBS/DMEM # 1.5% Seaplaque
agarose was added.
Infection was detected by staining
for NS5A as previously described.
To monitor viral
transmission in the presence of nAbs, anti-E2 C1 mAb
was added at a concentration (10
g/mL) able to neutral-
ize $90% infectivity, and media were replenished with
3% FBS/DMEM containing Ab every 24 hours.
quantify the infectivity of cell-free virus, extracellular me-
dia were collected and allowed to infect naı¨ve Huh-7.5
cells. Infection was quantiﬁed by the enumeration of
cells 48 hours post infection (pi.) and was deﬁned
as the number of infected cells or infectious units per
Proliferation Assay. Cells were labeled with 5 mM
carboxyﬂuorescein diacetate succinimidyl ester (CFSE;
CellTrace, Invitrogen) and seeded at the densities listed
previously. Labeled cells were infected with J6/JFH or
JFH and 24, 48, or 72 hours pi. were collected by
trypsinization and stained for NS5A, and the doubling
time for infected and noninfected cells was determined by
Infectious Center Assay. Huh-7.5 cells were infected
with J6/JFH or JFH at a multiplicity of infection of 0.01
and 96 hours pi. (producer cells) were mixed with naı¨ve
target cells labeled with 5
diacetate (CMFDA; CellTracker, Invitrogen). Producers
were incubated for 15 minutes at 37°C with Abs and
seeded with targets at a deﬁned ratio (see the legends) at
high density. nAbs were added at a minimum concentra-
tion 10 times greater than the deﬁned concentration that
inhibited 90% of infection (C1 and DEN3 at 10
NC2, P68, and P70 at 100
g/mL). After 24 hours, ex-
tracellular media were collected and quantiﬁed for infec-
tious virus as detailed previously. The cells were
18 TIMPE ET AL. HEPATOLOGY, January 2008
permeabilized with 1% paraformaldehyde/0.1% saponin,
stained for NS5A, and analyzed by ﬂow cytometry or
Routes of HCV Transmission. HCV infection of the
human hepatoma Huh-7.5 cell line results in focal areas
of infected cells expressing virally encoded structural
(core) and nonstructural (NS5A) proteins (Fig. 1), which
suggest localized viral transmission. A high degree of vari-
ability in the size of infected foci was noted, ranging from
1 to $200 infected cells. Because increased contact be-
tween target cells may potentiate viral transmission, we
examined the relationship between cell seeding density
and focal size. Infected cells were visualized by expression
of NS5A, and foci were partitioned into three groups on
the basis of size: fewer than 10, 10-50, and more than 50
infected cells. The proportion of large foci increased with
cell seeding density for both JFH and J6/JFH, and this
suggests that a high cell density facilitates viral transmis-
sion to neighboring cells. All subsequent assays to moni-
tor virus transmission were conducted at a high cell
To assess the role of cell division in viral transmission,
we quantiﬁed the proliferation time of naı¨ve and JFH-
infected Huh-7.5 by labeling cells with CFSE and mea-
suring proliferation over 72 hours. Under high-density
culture conditions, the mean doubling time for unin-
fected cells was 32 hours versus 34 hours for infected cells.
It is therefore unlikely that the large foci observed after 72
hours could derive simply from the division of infected
To address whether the development of foci requires
cell-free virus infection of naı¨ve cells, JFH-infected cells
were cultured in a semisolid medium containing agarose,
which limits the diffusion of virus particles. Inclusion of
an agarose overlay did not abrogate viral transmission but
resulted in compact foci reminiscent of plaques (Fig. 1D).
In contrast, infection of cells cultured in media alone
resulted in the development of medium to large foci sur-
rounded by smaller foci, or satellites (Fig. 1C). Inclusion
of an agarose overlay increased the proportion of large foci
at a high cell density from 26% to 70% for JFH, with a
concomitant decrease in small foci (&10; Fig. 1F). Paral-
lel assays studying J6/JFH-infected cells gave comparable
results (not shown). Thus, inclusion of an agarose overlay
to restrict cell-free virus transmission promotes localized
viral spread at high cell density, resulting in a greater
proportion of large foci.
The agarose overlay method has been used by virolo-
gists for many years. However, it is difﬁcult to assess its
effectiveness in limiting cell-free virus spread. In contrast,
inclusion of Abs in the extracellular media to inhibit viral
infectivity can be readily quantiﬁed. To monitor the level
of infectious virus released from JFH-infected cells, the
number of infected cells and the infectivity of cell-free
virus were quantiﬁed over a 72-hour period. Infectious
virus was readily detected between 48 and 72 hours pi.
However, the level of infectious virus released per infected
cell was low (approximately 1.0 IU per 5 infected cells;
Fig. 2B). JFH transmission in the presence or absence of
extracellular nAbs was assessed by the determination of
the frequency of infected cells and the infectivity of cell-
free virus at 72 hours pi. A concentration of anti-E2 (C1,
g/mL) capable of neutralizing JFH infectivity (Sup-
plementary Fig. 1) or a control anti-dengue (DEN3) mAb
Fig. 1. Formation of HCV foci: effects of cell density and agarose
overlay. Huh-7.5 cells were plated at low (gray), standard (white), or high
(black) seeding densities and infected with JFH in the presence or
absence of a 1.5% agarose overlay. At 72 hours pi., infected foci were
visualized by the detection of (A) core or (B,C) NS5A in cultures
maintained in media alone or (D) agarose overlay. The scale bar is 100
m. The number of infected cells within a focus were enumerated and
classiﬁed into 3 groups: fewer than 10, 10-50, and more than 50 cells
per focus. Fifty foci were counted at each seeding density. The percent-
age of the size of foci at different cell seeding densities in (E) the
absence of agarose and (F) the presence of agarose is shown. Cell
density (P & 0.0085,
correction for multiple testing) and agarose
signiﬁcantly increase the JFH focal size (P & 0.001,
HEPATOLOGY, Vol. 47, No. 1, 2008 TIMPE ET AL. 19
was added to the infected cultures at 8 hours pi. and
replenished every 24 hours to maintain bioactivity. C1
neutralized extracellular virus infectivity by 95% in com-
parison with untreated or DEN3-treated cells (Fig. 2C),
yet the relative frequency of infected cells in the C1-
treated culture was 43% versus 99% in the presence of
DEN3 (Fig. 2D). Infected foci in the presence of nAbs
exhibited fewer satellite colonies than untreated cultures,
and this is consistent with the effect(s) of agarose. In sum-
mary, these results show that HCV can transmit to naı¨ve
target cells in the presence of an agarose overlay or nAb,
suggesting that cell-cell routes of HCV transmission exist.
Cell-Cell HCV Transmission. To study cell-cell
transmission of infectivity, we developed an assay where
HCV-infected (producer) cells are cultured with ﬂuores-
cently labeled naı¨ve (target) cells in the presence of Abs
that neutralize the infectivity of cell-free virus. Labeling
the target cells allows one to discriminate between infec-
tion of naı¨ve cells and division of infected producers.
JFH-infected cells were seeded at high density in the pres-
ence or absence of nAbs for 15 minutes prior to the addi-
tion of labeled targets. Twenty-four hours later, the cells
were assayed for NS5A expression by indirect immuno-
ﬂuorescence, and extracellular media were quantiﬁed for
infectivity. Cell-free infectivity was reduced by $95% in
the presence of Abs. Figure 3 depicts the green-labeled
target cells and infected NS5A
producers indirectly la
beled red. Viral transmission from a producer to a target
cell results in a CMFDA-labeled green cell expressing
NS5A and costaining orange. HCV transmits to target
cells in the presence of C1 or puriﬁed Abs from two HCV-
infected patients (P68 and P70) in a comparable fashion
to untreated or control Ab–treated cultures (Fig. 3).
Thus, a $95% reduction in the infectivity of cell-free
virus has minimal effect(s) on the localized spread of
To obtain a quantitative analysis of viral transmission
in the presence of nAbs, cocultured cells were stained for
NS5A and analyzed by ﬂow cytometry. The upper left
quadrant shows the frequency of infected producers (red),
the upper right shows the frequency of virus-infected na-
ı¨ve targets (orange), and the lower right shows the fre-
quency of uninfected targets (green). JFH-infected targets
were detected at frequencies between 27% and 31% of
untreated or DEN3/NC2 control Ab–treated cultures
(Fig. 4A). In the presence of C1, P68, or p70 nAbs (Fig.
4B), the frequency of infected target cells ranged from
18% to 24%. The mean frequency of JFH-infected tar-
Fig. 3. HCV JFH transmits cell to cell. JFH-infected Huh-7.5 producers
were incubated for 15 minutes with (A) DMEM, (B) DMEM plus 10
g/mL DEN3, (C) DMEM plus 10
g/mL C1, or (D) DMEM plus 100
g/mL P70. The producers were cultured with naı¨ve CMFDA-labeled
Huh-7.5 targets (green). After 24 hours, infected cells were detected by
staining for NS5A (Alexa 594, red). Newly infected target cells, positive
for both NS5A and CMFDA, appear orange. The scale bar is 50
infectivity of cell-free virus in the presence of C1 or P70 was reduced by
$95% in comparison with DEN3 or untreated cultures.
Fig. 2. HCV JFH transmission occurs in the presence of an nAb.
Huh-7.5 cells were seeded at high density and infected for 1 hour at
37°C, and unbound virus was removed. (A) Cells were ﬁxed at 24, 48,
and 72 hours pi., and NS5A
cells were quantiﬁed. (B) The infectivity of
cell-free virus is expressed as the number of NS5A-positive cells or
infected units per milliliter. (C) The infectivity of cell-free JFH released
from control Ab DEN3–treated (white) or nAb C1–treated (black) cultures
is expressed as a percentage of the untreated culture (gray). (D) The
relative number of JFH-infected cells at 72 hours pi. in DEN3-treated
(white) or C1-treated (black) cultures is expressed as a percentage of the
untreated culture (gray). Infectivity for Ab-treated cells is expressed with
respect to untreated cells. Error bars represent the standard error of the
mean of 4 replicates and reﬂect the results from 4 independent exper-
20 TIMPE ET AL. HEPATOLOGY, January 2008
gets from three independent experiments is annotated
above each plot. In all cases, the nAbs (C1, P68, and P70)
reduced infectivity of cell-free virus by $95% (Fig. 4B)
with minimal effect(s) on cell-cell transfer of viral infec-
tivity. Increasing the concentration of C1 in the extracel-
lular media had no additional effect on the efﬁciency of
viral transfer (Supplementary Fig. 2). To ascertain that
cell-cell transmitted virus was not a variant resistant to the
nAbs under testing, infected cells were collected, and in-
tracellular virus was released by three rounds of rapid
freezing and thawing and tested for infectivity in the pres-
ence and absence of the selecting Ab. In all cases, the
intracellular virus remained sensitive to the neutralizing
effects of the Abs (Fig. 4C), and this indicated that viral
spread was not mediated by Ab-resistant variants. In sum-
mary, these data suggest that direct cell-cell transfer is an
efﬁcient route for HCV transmission between cells cul-
tured at high density.
Receptor Dependency of HCV Cell-Cell Transmis-
sion. To ascertain the role of CD81 in cell-free and cell-
cell infection, we evaluated the ability of an anti-CD81
mAb, M38 or sCD81, to inhibit HCV infection via these
two routes. M38 and sCD81 inhibited $90% of cell-free
infection of Huh-7.5 and reduced cell-cell transmission
by 43% and 15%, respectively (Fig. 5A,B). We investi-
gated whether the CD81-negative hepatoma cell line
HepG2 could be infected via cell-free or cell-cell infection
routes. HepG2 cells failed to support cell-free HCVcc
infection (Fig. 5D). However, coculturing of JFH-in-
fected producers with labeled HepG2 for 48 hours re-
sulted in 5.5% of HepG2 becoming infected versus 0.3%
for the nonpermissive Hela cell line (Fig. 5E). These data
conﬁrm that CD81-independent routes of cell-cell trans-
Fig. 5. The role of CD81 in HCV cell-cell transmission. Anti-CD81 M38
g/mL) and sCD81 (10
g/mL) inhibition of (A) cell-free JFH
infection, where infectivity is expressed as the number of NS5A-positive
cells or infected units per milliliter, and (B) cell-cell JFH infection, where
producer and targets were mixed in a 1:4 ratio for 24 hours. The mean
number of infected Huh-7.5 targets [# standard error of the mean
(SEM)] in the presence of p70, plus or minus M38 and sCD81, is shown
and is representative of 3 independent experiments. (C) CD81 cell
surface expression was monitored by ﬂow cytometry, and the data are
expressed as the mean ﬂuorescence intensity (MFI). (D) JFH cell-free
infection of targets. Infectivity is shown with respect to Huh-7.5, where
the error bars represent the SEM of three replicates. (E) JFH cell-cell
infection: producer and Huh-7.5, HepG2, and HeLa target cells were
mixed in a 1:4 ratio for 48 hours in the presence of p70. The mean
number of infected targets (#SEM) is shown and is representative of 3
Fig. 4. Quantitation of HCV cell-cell transmission. (A) JFH-infected
Huh-7.5 producers were cocultured with CMFDA-labeled targets at a ratio
of 1:4 in the presence of the listed Abs. After 24 hours, the cells were
ﬁxed, stained for NS5A (R-phycoerythrin) and infected cells quantiﬁed by
ﬂow cytometry. In each panel, the lower quadrants contain uninfected
cells; the upper left represents infected producers, and the upper right
represents newly infected targets. The mean number of infected targets
[# standard error of the mean (SEM)] from three independent experi-
ments is shown above each panel. (B) The infectivity of cell-free JFH
released from Ab-treated cultures is expressed as a percentage of the
untreated culture. The mean infection of triplicate wells (#SEM) with
respect to the untreated control is shown. (C) To conﬁrm that JFH in the
infected cultures was sensitive to nAbs, cell lysates were generated by
three freeze/thaw cycles and used to inoculate Huh-7.5 cells in the
presence (gray) or absence (white) of the appropriate nAb. The infectivity
of three replicates (#SEM) was measured and expressed with respect to
HEPATOLOGY, Vol. 47, No. 1, 2008 TIMPE ET AL. 21
To assess the role of CLDN1 in cell-cell transmission,
we studied cell-free and cell-cell infection of the human
embryonal 293T kidney cell line before (CD81
) and after transduction (CD81
express CLDN1. CLDN1 expression levels were con-
ﬁrmed by ﬂow cytometry and receptor activity by
HCVpp and HCVcc infection (Fig. 6A-C). Expression of
CLDN1 in 293T cells increased cell-cell transmission
5-fold in comparison with 293T, which gave background
levels of infection comparable to that seen with Hela cells
(Fig. 6D). In contrast, 293T-CLDN1 cells were poor
targets for JFH cell-free virus infection (Fig. 6C). In sum-
mary, these data support a model in which cell-cell trans-
mission is dependent on CLDN1 expression and CD81-
dependent and independent routes exist.
In this study, we demonstrate that HCV can transmit
to naı¨ve cells in the presence of agarose or Abs that limit or
neutralize cell-free virus infectivity. Intracellular sources
of virus remain sensitive to the neutralizing activity of
Abs, and this conﬁrms that the transmitting viruses are
not resistant to the nAbs used. The frequency of infected
naı¨ve target cells in the infectious center assay was mini-
mally affected by the inhibition of extracellular routes of
virus transmission. These data suggest that HCV can
transmit by at least two routes in vitro: cell-free virus
infection of naı¨ve targets and direct transfer between cells.
The latter route offers a mechanism to evade nAbs that
may partially explain the ineffectiveness of Abs in control-
ling HCV replication during the chronic phase of dis-
in addition to more conventional genetic escape
If such routes of transmission occur in vivo,
one may question whether therapeutic vaccination to
or immunoprophylaxis will control persis-
tent HCV replication.
To address the receptor dependency of HCV transmis-
sion between infected and naı¨ve cells, we used cell lines
lacking CD81 or CLDN1 expression as targets for cell-
free and cell-cell transfer of infection. CD81-negative
HepG2 cells failed to support cell-free HCVcc infection
but were infected after coculturing with JFH-infected
producers (Fig. 5). This observation, alongside the partial
inhibition of JFH cell-cell transmission by sCD81 and
anti-CD81 to Huh-7.5 targets, suggests that CD81-de-
pendent and CD81-independent routes of cell-cell trans-
mission occur. The recent discovery that the tight
junction protein CLDN1 is an essential factor allowing
HCV entry into cells prompted us to study its role in
Expression of CLDN1 in 293T
target cells conferred JFH infection via cell-cell transmis-
sion (Fig. 6), and this suggests that CLDN1 expression in
the target cell is essential for cell-cell transfer of infectivity.
The lower level of JFH cell-cell transmission to HepG2
and 293T-CLDN1 compared to Huh-7.5 cells most
likely reﬂects the reduced ability of HepG2 and 293T
cells to support HCV RNA replication.
We noted considerable variability in the size of infected
foci, with some producer cells failing to transmit virus to
naı¨ve target cells (Fig. 3). One explanation may reﬂect the
heterogeneous expression of CD81 and CLDN1 in Huh-
7.5 targets. An alternative explanation may be the inher-
ent variability of Huh-7 in supporting HCV RNA
Indeed, close inspection of foci in the
infectious center assay allows one to track the transfer of
HCV infectivity from producer to labeled targets. NS5A
or core expression in the target cell is variable and does not
associate with proximity to producer cells, which most
likely reﬂects variation in the target’s ability to support
HCV RNA replication (Fig. 3).
Target cell density affects HCV transmission, with an
increased proportion of large foci at high density (Fig. 1).
HCVpp entry into Huh-7.5 cells is enhanced when cells
are seeded at higher density, and this supports a model in
which cell-cell contact facilitates the expression or com-
plex formation of host cell molecules required for HCV
entry (A.S., unpublished observations, 2007). It is inter-
Fig. 6. The role of CLDN1 in HCV cell-cell transmission. (A) Target cell
expression of CLDN1 was monitored by ﬂow cytometry, and data are
expressed as the mean ﬂuorescence intensity (MFI). (B) HCVpp and
murine leukemia virus pseudoparticle (MLVpp) infection of targets. Pseu-
doparticles encode a luciferase reporter, and data are presented as the
speciﬁc infectivity (SI), where the HCVpp (gray) or MLVpp (white) signal
with respect to a pseudoparticle lacking envelope is determined from
three replicate infections [# standard error of the mean (SEM)]. (C) JFH
cell-free infection of targets. Infectivity is shown with respect to Huh-7.5,
and the error bars represent the SEM of three replicates. (D) JFH cell-cell
infection: producer and 293T, 293T-CLDN1, and HeLa target cells were
mixed in a 1:1 ratio for 48 hours in the presence of p70. The mean
number of infected targets (#SEM) is shown and is representative of 3
22 TIMPE ET AL. HEPATOLOGY, January 2008
esting to speculate on the physiological signiﬁcance of
these results. The principal site of HCV replication in vivo
is thought to be hepatocytes within the liver, which form
polarized sheets where the cells are at high density (2-
3.0 " 10
of liver tissue).
HCV RNA has been reported to replicate more efﬁciently
in actively proliferating cells, demonstrating reduced rep-
lication in cells at high density.
Because the majority
of hepatocytes within the liver are not proliferating and
are arrested in G
, these data suggest a delicate balance
between the cell requirements that are optimal for HCV
entry and transmission versus those required for efﬁcient
viral RNA replication.
Cell-cell spread of viruses in solid tissues is a complex
and poorly understood process.
The alphaherpes viruses
replicate in polarized cells, epithelial cells, and neurons
and mimic intracellular sorting pathways to promote in-
fection of adjacent cells.
virus and HTLV-I infect T cells that form immunological
synapses with cells of the immune system. Both viruses
appear to use these pathways to form viral synapses to
facilitate the transfer of infectivity between immune cell
It is noteworthy that JFH-infected Huh-7.5 cells re-
lease low levels of infectious virus (Fig. 2C) that may favor
cell-cell transfer of viral infectivity in vitro. At the present
time, we can only speculate on the mechanism(s) of HCV
transmission in vivo both within and between hosts. Iden-
tiﬁcation of HCV-infected cells within the liver has been
difﬁcult to demonstrate with conﬂicting reports, and this
makes viral production rates difﬁcult to ascertain.
recent report by Gale and colleagues
areas of infected cells within the liver that were consistent
with our in vitro observations.
Plasma from HCV-in-
fected individuals has been reported to infect chimpan-
and mice bearing chimeric human livers,
suggests that transmission between hosts is most likely
mediated via cell-free virus. Our data support a model in
which HCV may transmit within the liver by multiple
routes, including cell-free virus and direct cell-cell trans-
fer, with the latter offering a novel route for evading nAb
responses. These data may partly explain the inability of
nAbs to control HCV replication during the chronic
phase of disease and raise concerns over the effectiveness
of therapeutic vaccination targeting the humoral immune
response and of antiviral agents targeting CD81-depen-
dent routes of infection.
Acknowledgment: We thank Takaji Wakita for JFH;
Charles Rice for J6/JFH, 9E10, and Huh-7.5 cells; Den-
nis Burton for C1; John McLauchlan for JM122; and
Fedor Berditchevski for M38.
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