JOURNAL OF VIROLOGY, June 2009, p. 5477–5484
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 11
Residues in a Highly Conserved Claudin-1 Motif Are Required for
Hepatitis C Virus Entry and Mediate the Formation of
Lisa Cukierman, Laurent Meertens, Claire Bertaux, Francis Kajumo, and Tatjana Dragic*
Albert Einstein College of Medicine, Department of Microbiology and Immunology, 1300 Morris Park Avenue,
Bronx, New York 10461
Received 28 October 2008/Accepted 13 March 2009
Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage
entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry,
we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important.
However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV
isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin
motif W30-GLW51-C54-C64. Alanine substitutions of these residues did not impair claudin-1 cell surface
expression or lateral protein interactions within the plasma membrane, including claudin-1–claudin-1 and
claudin-1–CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our
observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane
domains that are amenable to HCV entry.
Hepatitis C virus (HCV) is a major human pathogen that
affects approximately 3% of the global population, leading to
cirrhosis and hepatocellular carcinoma in chronically infected
individuals (5, 23, 42). Hepatocytes are the major target cells of
HCV (11), and entry follows a complex cascade of interactions
with several cellular factors (6, 8, 12, 17). Infectious viral par-
ticles are associated with lipoproteins and initially attach to
target cells via glycosaminoglycans and the low-density lipo-
protein receptor (1, 7, 31). These interactions are followed by
direct binding of the E2 envelope glycoprotein to the scavenger
receptor class B type I (SR-B1) and then to the CD81 tet-
raspanin (14, 15, 33, 36). Early studies showed that CD81
and SR-B1 were necessary but not sufficient for HCV entry,
and claudin-1 was discovered to be a requisite HCV entry
cofactor that appears to act at a very late stage of the
Claudin-1 is a member of the claudin protein family that
participates in the formation of tight junctions between adja-
cent cells (25, 30, 37). Tight junctions regulate the paracellular
transport of solutes, water, and ions and also generate apical-
basal cell polarity (25, 37). In the liver, the apical surfaces of
hepatocytes form bile canaliculi, whereas the basolateral sur-
faces face the underside of the endothelial layer that lines liver
sinusoids. Claudin-1 is highly expressed in tight junctions
formed by liver hepatocytes as well as on all hepatoma cell
lines that are permissive to HCV entry (18, 24, 28). Impor-
tantly, nonhepatic cell lines that are engineered to express
claudin-1 become permissive to HCV entry (18). Claudin-6
and -9 are two other members of the human claudin family that
enable HCV entry into nonpermissive cells (28, 43).
The precise role of claudin-1 in HCV entry remains to be
determined. A direct interaction between claudins and HCV
particles or soluble E2 envelope glycoprotein has not been
demonstrated (18; T. Dragic, unpublished data). It is possible
that claudin-1 interacts with HCV entry receptors SR-B1 or
CD81, thereby modulating their ability to bind to E2. Alterna-
tively, claudin-1 may ferry the receptor-virus complex to fu-
sion-permissive intracellular compartments. Recent studies
show that claudin-1 colocalizes with the CD81 tetraspanin at
the cell surface of permissive cell lines (22, 34, 41). With
respect to nonpermissive cells, one group observed that clau-
din-1 was predominantly intracellular (41), whereas another
reported associations of claudin-1 and CD81 at the cell sur-
face, similar to what is observed in permissive cells (22).
Claudins comprise four transmembrane domains along with
two extracellular loops and two cytoplasmic domains (19, 20,
25, 30, 37). The first extracellular loop (ECL1) participates in
pore formation and influences paracellular charge selectivity
(25, 37). It has been shown that the ECL1 of claudin-1 is
required for HCV entry (18). All human claudins comprise a
highly conserved motif, W30-GLW51-C54-C64, in the crown of
ECL1 (25, 37). The exact function of this domain is unknown,
and we hypothesized that it is important for HCV entry. The
second extracellular loop is required for the holding function
and oligomerization of the protein (25). Claudin-1 also com-
prises various signaling domains and a PDZ binding motif in
the intracellular C terminus that binds ZO-1, another major
component of tight junctions (30, 32, 37). We further hypoth-
esized that some of these domains may play a role in HCV
To understand the role of claudin-1 in HCV infection, we
developed a mutagenesis strategy targeting the putative sites
ylation. The functionality of these domains has been described
by others (4, 16, 25, 35, 37, 40). We also mutagenized charged
* Corresponding author. Mailing address: Albert Einstein College
of Medicine, Department of Microbiology and Immunology, 1300
Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-3282. Fax:
(718) 430-8711. E-mail: firstname.lastname@example.org.
?Published ahead of print on 18 March 2009.
and bulky residues in ECL1, including all six residues within
the highly conserved motif W30-GLW51-C54-C64. None of the
intracellular domains were found to affect HCV entry. How-
ever, we identified seven residues in ECL1 that are critical for
entry mediated by envelope glycoproteins derived from several
HCV subtypes, including all six residues of the conserved mo-
tif. These mutants were still expressed at the cell surface and
able to form lateral homophilic interactions within the plasma
membrane as well as to engage in lateral interactions with
CD81. In contrast, they no longer engaged in homophilic trans
interactions at cell-cell contacts. We conclude that the highly
conserved motif W30-GLW51-C54-C64of claudin-1 is important
for HCV entry into target cells and participates in the forma-
tion of cell-cell contacts.
MATERIALS AND METHODS
Cells and mutagenesis. The human kidney endothelial (HEK) cell line was
obtained through the American Type Culture Collection. Huh-7 and Huh-7.5
cells were provided by C. Rice (Rockefeller University, NY) and H1H cells by R.
Chowdhurry (Albert Einstein College of Medicine, Bronx, NY). Mutagenesis of
the claudin-1-coding sequence was performed using QuikChange (Stratagene)
according to the manufacturer’s instructions, and the sequence was subcloned
into the pQCXIN retroviral expression vector (Clontech) using NotI and BamHI
restrictions enzyme sites. Vesicular stomatitis virus pseudoparticles (VSVpp) for
the delivery of pQCXIN-claudin-1 were generated in HEK cells and used to
transduce HEK or H1H cells, which were selected with G418 (1 mg/ml; Invitro-
gen) for 1 to 2 weeks, as previously described by us (28).
Generation of HCVpp and infection of target cells. Pseudoparticles were made
by cotransfecting the NLenv-luc? vector with a vector encoding HCV, VSV, or
envelope glycoproteins, as previously described by us (10, 14, 28, 29). HCV
envelope glycoprotein sequences derived from subtypes 1a, 1b, 2b, 3a, 4, and 5
were also described by us (28). Viral titers were determined by infecting Huh-7
cells with the viral stocks and measuring luciferase activity in cell lysates at 48 h
postinfection. Similar luciferase titers were then used to infect HEK or H1H cells
stably expressing wild-type or mutant claudin-1. The percentage of entry of HCV
pseudoparticles (HCVpp) was calculated as a percentage of entry into cells
expressing the wild-type claudin-1 protein.
Immunoblotting of claudin-1 expressed in transduced cells. Proteins in the
lysates of HEK or H1H cells (3 ? 106) were separated by 14% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (Invitrogen) and transferred to pure
nitrocellulose membranes (Bio-Rad). These were probed with a JAY.8 rabbit
polyclonal antibody against claudin-1 (1:500; Invitrogen) or a rabbit polyclonal
antibody against ?-tubulin (1:500; Santa Cruz Biotechnology). Staining was re-
vealed with a horseradish peroxidase (HRP)-conjugated donkey anti-rabbit an-
tibody (1:10,000; Amersham Biosciences) and developed using Western Light-
ning Chemiluminescence Reagent Plus (PerkinElmer).
Biotinylation of cell surface proteins and detection of claudin-1. HEK cells
(8 ? 106) expressing mutant or wild-type claudin-1 were biotinylated at 4°C for
30 min with EZ-Link Sulfo-NHS-LC-biotin (0.5 mg/ml; Pierce) according to the
manufacturer’s instructions. The reaction was quenched by washing with 10 mM
glycine (Sigma) in phosphate-buffered saline. Radioimmunoprecipitation assay
buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% SDS, 1% NP-40, 1%
sodium deoxycholate, 1 mM EDTA, 2 mM Na3VO4and Pierce protease inhib-
itor cocktail) was used to lyse the cells, and biotinylated proteins were pulled
down with streptavidin agarose resin (Pierce). Proteins were stripped off the
resin by heating for 5 min at 95°C in radioimmunoprecipitation assay buffer, and
Western blotting of proteins was performed as described in the previous section.
Golgin-97 was detected with a rabbit polyclonal antibody (1:50; Abcam), fol-
lowed by HRP-conjugated donkey anti-rabbit antibody (1:10,000; Amersham
BiFC. The biomolecular fluorescence complementation (BiFC) assay was
adapted from that described by Yang et al. (41). Briefly, sequences encoding
either the C- or N-terminal moiety of the Venus fluorescent protein (termed vc
and vn, respectively) were appended to the 3? ends of wild-type or mutant
claudin-1 or to CD81-coding sequences. Plasmids expressing complementary
chimeric proteins were transiently transfected in a 1:1 ratio into HEK cells (3 ?
106) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s
instructions. After 48 h, cells were harvested and analyzed by flow cytometry
(fluorescein isothiocyanate, FACSCalibur). Background fluorescence was deter-
mined by cotransfecting wild-type claudin-1 appended with vn or CD81 ap-
pended with vc and pQCXIN vectors encoding only vc or vn, respectively.
Immunofluorescence. HEK cells stably expressing wild-type or mutant clau-
din-1 or Huh-7 cells were incubated on Lab-Tek chambers (Fisher) at 70%
confluence, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton
X-100, treated with NaBH4(1 mg/ml), and blocked with 0.1% Tween–2% bovine
serum albumin–2% normal donkey serum. Cells were costained for detection of
claudin-1 or mutants using a JAY.8 rabbit polyclonal antibody (1:100; Invitro-
gen) and for ZO-1 using a ZO-1-1A12 monoclonal mouse antibody (1:100;
Invitrogen). As secondary antibodies we used Cy3-conjugated donkey anti-rabbit
immunoglobulin G (IgG) or Cy2-conjugated donkey anti-mouse IgG antibody
(1:500; Jackson Immunoresearch). Nuclei were stained with Hoechst 33258 (In-
vitrogen). Images were generated using a Leica AOBS laser scanning confocal
microscope (63? objective). Claudin-1 expression in Huh-7 cells was analyzed
using the same protocol.
Intracellular claudin-1 residues are not required for HCV
entry into target cells. Recently identified as a cofactor for
HCV replication, claudin-1 is known to act at a late stage of
entry, but its exact function remains unclear. To better under-
stand the role of claudin-1 in HCV entry, we performed ala-
nine scanning mutagenesis of single residues predicted to be
involved in various putative functions (4, 16, 25, 35, 37, 40)
(Fig. 1). Alanines were introduced in place of C-terminal do-
main residues belonging to an internalization motif (Y193A,
P194A, Y199A, or P200A) or a phosphorylation site (T195A).
Cysteines were alanine substituted to study the role of palmi-
toylation sites (C104, C107, C183A, C184A, or C186A). We
then generated stable HEK cell lines expressing the various
claudin mutants and tested their ability to be infected by
HCVpp bearing the envelope glycoproteins of the H77 (1a)
FIG. 1. Two-dimensional structure of claudin-1. Claudin-1 is a
four-transmembrane-domain protein with two extracellular loops and
two cytoplasmic domains. Human claudin-1 is not polymorphic. Res-
idues that we mutagenized are indicated in ovals. The residues in bold
ovals, W30-GLW51-C54-C64, belong to the highly conserved motif of
claudin proteins. The percentages of conservation of these residues
within the 24 human claudins are indicated in the top right panel
(NCBI Database and ClustalW).
5478CUKIERMAN ET AL.J. VIROL.
isolate, as previously described by us (10, 14, 28). None of the
mutations described in this section significantly affected
HCVpp entry into HEK cells (Fig. 2A). We confirmed these
results using the human hepatoma cell line H1H, which ex-
presses very low levels of endogenous claudin-1 and is there-
fore not permissive to HCV entry (Fig. 2B). Based on our
observations, we concluded that intracellular functional do-
mains of claudin-1 are not important for HCV entry.
Residues in ECL1, including the W30-GLW51-C54-C64highly
conserved motif, are required for HCV entry. Next we explored
the role of residues in ECL1 of claudin-1, with special empha-
sis on its N-terminal half, which was previously shown to be
important for HCV entry (Fig. 1) (18). We introduced single
alanine substitutions at positions occupied by bulky, charged,
or polar residues, including R31A, I32A, Y33A, Y35A, D38A,
N39A, T42A, Q44A, A45G, M52A, S53A, K65A, and N72A, a
potential glycosylation site (Fig. 1). We also mutated each of
the residues in the highly conserved motif W30-GLW51-C54-
C64(Fig. 1). HEK cells stably expressing wild-type or mutant
claudin-1 were infected with HCVpp bearing the envelope
glycoproteins of isolate H77 (1a). We confirmed that the pre-
viously described residue I32 is important for entry (18) and
also showed that the proximal D38 residue was equally impor-
tant (Fig. 3A). The following ECL1 mutants, however, had no
significant effect on HCVpp entry: R31A, Y33A, Y35A, N39A,
T42A, Q44A, A45G, M52A, S53A, K65A, and N72A (Fig. 3).
In contrast, all six residues within the conserved motif, includ-
ing W30-GLW51-C54-C64, were critical, because their alanine
substitutions enabled only ?15% of the HCVpp entry ob-
served for the wild-type protein (Fig. 3B). We also tested the
ability of human T-cell leukemia virus type 1 pseudoparticles
and VSVpp to enter the various HEK-claudin-1 derivatives
and found no effect of the claudin-1 mutants, indicating that
the observed effects were specific for HCVpp (data not
Critical claudin-1 residues are broadly important for entry
of various HCV subtypes. In order to determine whether ECL1
residues that were found to impair HCVpp 1a entry were also
important for entry mediated by the envelope glycoproteins of
other HCV subtypes, we extended our studies to include
HCVpp generated with the envelope glycoproteins from pa-
tient isolates previously described by us (28). We found that
the residues that we identified as being important for entry of
the H77 (1a) isolate were also important for entry of isolates
belonging to subtypes 1b, 2b, 3a, 4, and 5 (Fig. 4). There were
some differences in the degree to which entry mediated by the
various envelope glycoproteins was affected, such that each
isolate represented a unique signature pattern of residue us-
age. For example, HCVpp 1b entry was most severely sup-
pressed by substitutions at positions 30, 32, 50, 54, and 64;
HCVpp 2b entry was most sensitive to substitutions at posi-
tions 30, 49, 50, and 64; and HCVpp 3a entry was most sensi-
tive to the substitution at position 64 (Fig. 4). Together, our
FIG. 2. Intracellular claudin-1 residues do not affect HCVpp entry.
HEK cells (A) or H1H cells (B) were transduced to express wild-type
(wt) or mutant claudin-1, as indicated along the x axis. Cells were
infected with HCVpp subtype 1a (isolate H77), and luciferase activity
(relative light units) in cell lysates was measured at 48 h postinfection.
The percentage of HCVpp entry enabled by claudin-1 mutants was
calculated as a percentage of entry into cells expressing the wild-type
protein ? standard deviation. Values are averages from four indepen-
FIG. 3. Residues in ECL1 are critical for HCVpp entry. HEK cells
were transduced to stably express claudin-1 mutants of charged or
polar ECL1 residues (A) or conserved motif residues (B), as indicated
along the x axis, and infected with HCVpp subtype 1a (isolate H77).
Luciferase activity (relative light units) in cell lysates was measured at
48 h postinfection. The percentage of HCVpp entry enabled by clau-
din-1 mutants was calculated as a percentage of entry into cells ex-
pressing the wild-type (wt) protein ? standard deviation. Values are
averages from three independent experiments.
VOL. 83, 2009 CLAUDIN-1 MOTIF REQUIRED FOR HCV ENTRY5479
results confirm the importance of seven ECL1 residues in entry
of six HCV subtypes.
Claudin-1 mutants that are critical for HCVpp entry are
expressed at levels similar to those of the wild-type protein
and are on the cell surface. We performed experiments to
ascertain expression, transport, and folding of the various clau-
din-1 mutants no longer capable of enabling HCVpp entry.
Initially, lysates of HEK cells transformed and selected for
expression of wild-type or mutant claudin-1 were probed by
Western blotting using a rabbit anti-claudin-1 antibody specific
for the C-terminal domain of claudin-1 (Fig. 5A, top). The
various HEK cell populations were found to express similar
levels of the 22-kDa band corresponding to wild-type or mu-
tant claudin-1 protein. Equal amounts of lysates were depos-
ited in the wells as shown by the consistency of the beta-tubulin
control (Fig. 5A, bottom). Note that expression of claudin-1 in
transduced HEK cells is similar to its endogenous expression
levels in Huh-7 and Huh-7.5 hepatoma cells as well as primary
human hepatocytes (Fig. 5B).
We then performed experiments to establish whether mu-
tant claudin-1 proteins were transported to the cell surface,
like the wild-type protein. Because the only commercially
available anti-claudin-1 antibody is directed against an epitope
in the intracellular C terminus, detection of claudin-1 could
not be realized by flow cytometry. Consequently, we used a cell
surface biotinylation approach to purify and detect plasma
membrane-associated claudin-1. Briefly, whole cells were
treated with NHS-biotin and biotinylated proteins pulled out
with streptavidin-conjugated agarose. After elution and West-
ern blotting with the anti-claudin-1 antibody, we showed that
all of the mutants that were impaired for HCV entry were
FIG. 4. Entry of all HCV test isolates depends on the same ECL1
residues. HEK cells expressing claudin-1 mutants found to be impor-
tant for HCVpp subtype 1a entry were also tested for their ability to
enable entry of HCVpp generated with the envelope glycoproteins of
subtypes 1b, 2b, 3a, 4, and 5, as indicated. Similar luciferase titers were
used for the various HCVpp, ranging from 10,000 to 100,000 relative
light units, which is within the linear range of the assay. The percentage
of HCVpp entry enabled by claudin-1 mutants was calculated as a
percentage of entry into cells expressing the wild-type (wt) protein ?
standard deviation. Values are averages from five independent exper-
FIG. 5. Claudin-1 mutants that abrogate HCV entry are expressed
similarly to wild-type claudin-1 and are transported to the cell surface.
(A) Lysates of HEK cells transduced with wild-type claudin-1 (wt CL1)
or critical mutants were analyzed by Western blotting. Membranes
were probed with antibodies against claudin-1 or ?-tubulin, as an
internal control, followed by a secondary HRP-conjugated donkey
anti-rabbit IgG antibody. (B) Lysates of HEK cells transduced with
wild-type claudin-1, Huh-7 cells, Huh-7.5 cells or primary human hepa-
tocytes (PHH) were analyzed by Western blotting using antibodies
against claudin-1 or ?-tubulin, as an internal control. (C) HEK cells
stably expressing wild-type claudin-1 or critical mutants were biotinyl-
ated or mock treated with buffer. Proteins were extracted with strepta-
vidin-conjugated agarose, and claudin-1 was detected by Western blot-
ting as for panel A. (D) Alternatively, golgin-97, an integral membrane
protein from the Golgi apparatus, was detected with an anti-golgin-97
monoclonal antibody in biotinylated or nonbiotinylated HEK cell
5480CUKIERMAN ET AL.J. VIROL.
transported to the cell surface, similar to the case for the
wild-type protein (Fig. 5C). To ascertain that only plasma
membrane-associated proteins were biotinylated and detected,
we also tested for the presence of an intracellular protein,
golgin-97, which is an integral membrane protein in the Golgi
apparatus. Only trace levels of golgin-97 were detected in the
biotinylated fractions, indicating that the majority of biotinyl-
ated claudin-1 is from the cell surface (Fig. 5D). Overall, our
data show robust expression of entry-impaired claudin-1 mu-
tants, similar to that of the wild-type protein. The lack of
function in HCVpp entry of our ECL1 mutants is not due to
changes in their expression levels or their ability to reach the
ECL1 mutations that impair HCVpp entry do not affect
protein interactions at the cell surface. We used a BiFC assay
to determine whether the claudin-1 mutations that impair
HCVpp entry affect claudin-1 lateral interactions with itself or
with CD81. The C termini of wild-type or mutant claudin-1
were appended with Venus fluorescent protein moieties, either
vn or vc. Similarly tagged versions of CD81 were also gener-
ated. In this assay, vn and vc are not able to fluoresce sepa-
rately. However, if two proteins each bearing one of the moi-
eties interact and bring the C and N termini within ?15 nm of
each other, a detectable fluorescence signal is generated by
complementation. Using flow cytometry to detect Venus fluo-
rescence, we showed that wild-type claudin-1 proteins interact
with each other, as do the mutants that were shown to be
impaired for HCV entry (Fig. 6). Though there is a slight
decrease in BiFC generated by the claudin-1 mutants com-
pared to the wild type, it is not statistically significant (P ?
0.6124 for I32 and P ? 0.5981 for W-GaW-C-C in an unpaired,
two-tailed t test). Moreover, we observed that mutations in the
conserved domain do not affect lateral interactions between
claudin-1 and CD81 as there was no significant difference be-
tween BiFC generated by coexpression of CD81 with wild-type
or mutant claudin-1 proteins (P ? 0.4437 for I32 and P ?
0.5981 for W-GaW-C-C in an unpaired, two-tailed t test) (Fig.
Mutations in the conserved motif that affect HCVpp entry
affect homophilic trans interactions at cell-cell contacts. Six of
the seven residues that we identified as being critical for
HCVpp entry belong to the highly conserved motif W30-
GLW51-C54-C64. Because this motif is conserved in all clau-
dins, we hypothesized that it is involved in the formation of
cell-cell contacts. We explored claudin-1 trans interactions by
studying the ability of wild-type and mutant proteins to colo-
calize with another component of tight junctions, ZO-1, at
cell-cell contacts. Based on immunofluorescence analyses, we
showed that wild-type claudin-1 as well as a mutant that does
not affect HCVpp entry localized at cell-cell contacts and co-
localized with ZO-1 (Fig. 7A and B). In contrast, mutants of
the conserved motif that impaired HCVpp entry were not
detected at cell-cell contacts, nor did they colocalize with ZO-1
(Fig. 7C and D and data not shown). Note that claudin-1 tends
to accumulate intracellularly in transduced HEK cells as well
as Huh-7 hepatoma cells (data not shown). We conclude that
the highly conserved motif W30-GLW51-C54-C64is required for
the formation of cell-cell contacts as well as for HCV entry.
CD81 and SR-B1 initially entered the HCV field as envelope
glycoprotein E2-binding proteins (14, 15, 33, 36). With the
development of the HCVpp and HCV cell culture assays, it
was demonstrated that these cell surface molecules are HCV
entry receptors (9, 14, 21, 26, 27, 38). At that time it also
became clear that SR-B1 and CD81 were necessary but insuf-
ficient to mediate HCV entry into target cells. Using a func-
tional cloning approach, Evans et al. identified the tight junc-
tion component claudin-1 as yet another, indispensable HCV
entry cofactor (18). Claudin-1 is expressed by all HCV-permis-
sive cells, and its expression in CD81?and SR-B1?cells ren-
ders them permissive to the virus. Follow-up studies by us and
others demonstrated that claudin-6 and -9, but not -2, -3, -4, -7,
-11, -12, -15, -17, and -23, could also fulfill the role of HCV
entry cofactor (28, 43).
In their study, Evans et al. (18) analyzed chimeras of clau-
din-1 and the closely related claudin-7, observing that the in-
tracellular domains of claudin-1 and claudin-7 were inter-
changeable vis-a-vis HCV entry. However, this did not address
whether functions associated with these domains were in fact
required. Using an alanine scanning mutagenesis strategy, we
investigated the necessity for various putative functional do-
mains of claudin-1 in HCV entry, including motifs for inter-
nalization, glycosylation, palmitoylation, and phosphorylation.
Here, we report that none of these putative functions of clau-
din-1 play a role in HCV entry, a surprising finding in light of
the observation that claudin-1 intervenes late in the process of
viral entry. Furthermore, our observations suggested that the
cofactor function of claudin-1 was strictly associated with cel-
lular functions fulfilled by its extracellular domain.
FIG. 6. Mutations of claudin-1 residues critical for HCV entry con-
tinue to engage in lateral interactions within the plasma membrane.
Equal quantities of vc- and vn-tagged constructs, indicated along the x
axis, were cotransfected into HEK cells, which were analyzed by flow
cytometry at 48 h posttransfection. Mean fluorescence intensity is
indicated along the y axis. Fluorescence signals generated by various
claudin-1 combinations were all significantly higher than the control
signal generated by claudin-1-vc and pQCXIN-vn (all P values were
?0.001 as determined by an unpaired, two-tailed t test). Similarly,
fluorescence signals generated between CD81 and various claudin-1
variants were significantly higher than the control (all P values were
?0.007). Values are averages plus or minus standard deviations from
five independent experiments.
VOL. 83, 2009CLAUDIN-1 MOTIF REQUIRED FOR HCV ENTRY5481
The requirement for specific residues in ECL1 was therefore
explored. In addition to previously identified residues I32 and
E48, we found seven amino acids that are critical for entry of
six HCV isolates belonging to subtype 1a, 1b, 2b, 3a, 4, or 5. Six
of the residues belong to the highly conserved motif (W30-
GLW51-C54-C64) (25, 37), and one (D38) is external to this
motif. We confirmed that the lack of function of our mutants
was not due to a defect in expression or protein transport to
the cell surface. Residues I32, D38, and E48 may be involved
in the passage of cations across tight junctions (2, 3, 13), but
this function of claudin-1 has not yet been directly linked to
HCV entry. The role of I32, D38, and E48 therefore remains
to be explored, and these residues may determine HCV spec-
ificity for claudin-1.
In this study, we focused on exploring the function of the
residues in the highly conserved, hydrophobic motif W30-
GLW51-C54-C64, which is found in all 24 members of the hu-
man claudin family. The two cysteines included in this motif, in
positions 54 and 64 in claudin-1, probably do not form a di-
sulfide bond, since we did not observe a size difference be-
tween reduced and nonreduced protein by Western blotting or
between alanine mutants and wild-type claudin-1 (T. Dragic,
unpublished data). However, the two cysteines have been de-
scribed to be important for tightness function as measured by
FIG. 7. Claudin-1 mutants with mutations in the highly conserved motif that impair HCV entry do not localize at cell-cell contacts. HEK cells
expressing wild-type (wt) claudin-1 (A); mutant M52A, which does not affect HCVpp entry (B), W-GaW-C-C (C), or W-GLW-C-a (D) were fixed
and immunostained for claudin-1 (red) and ZO-1 (green). Immunofluorescent images were captured with a Leica AOBS laser scanning confocal
microscope using a 63? objective. The merged images showing an overlay of wild-type claudin-1 or mutants and ZO-1 were generated using Image
J software. White arrows show the colocalization of claudin-1 and ZO.1 at cell-cell contacts.
5482 CUKIERMAN ET AL.J. VIROL.
transepithelial electrical resistance (25, 39). This led us to
speculate that the conserved motif is involved in the formation
of cell-cell contacts.
The ability of W30-GLW51-C54-C64mutants to polymerize
via lateral interactions (cis interaction) within the same mem-
brane and to participate in head-to-head interactions (trans
interaction) for adhesion of adjacent membranes was explored.
Using a BiFC assay, we showed that motif mutations do not
affect claudin-1–claudin-1 or claudin-1–CD81 lateral interac-
tions within the membrane. On the other hand, we observed by
confocal microscopy that mutants within the conserved motif
were not able to form homophilic trans interactions since they
did not localize at cell-cell contacts along with ZO-1. These
data suggested that residues important for HCV entry do not
affect cis interactions but do affect trans interactions required
for cell-cell adhesion. Thus, cell-cell contacts formed by clau-
din-1 may represent specialized membrane domains required
for HCV entry. Formation of these domains involves residues
in the highly conserved motif W30-GLW51-C54-C64.
Evans et al. (18) showed that claudin-1 intervenes at a very
late stage of HCV entry. We hypothesize that claudin-1 inter-
acts either with the HCV envelope glycoproteins or with one of
the other HCV entry cofactors. To date, there are no reports
of claudin-1–HCV envelope glycoprotein interactions, either
because claudin-1 interacts with E1, for which there is cur-
rently no binding assay, or because it interacts with a confor-
mation of E2 that is not reproduced by available models, in-
cluding soluble E2, HCVpp, or HCV cell culture. Based on our
data, we propose that the formation of cell-cell contacts by
residues in the highly conserved motif W30-GLW51-C54-C64is
necessary for the recruitment of the HCV-receptor complex to
membrane domains that are amenable to its internalization
and trafficking into fusion-permissive intracellular compart-
We thank Tianyi Wang for generously providing us with constructs
encoding the N- and C-terminal moieties of Venus fluorescence
This work was supported by NIH grant AI060390 and the Burroughs
Wellcome Fund, Investigators in Pathogenesis of Infectious Diseases.
This work was also supported in part by NIAID Centers for AIDS
Research grant AI051519 to Albert Einstein College of Medicine.
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