Evans, M. J. et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801-805.OpenURL

Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave, New York 10021, USA.
Nature (Impact Factor: 41.46). 05/2007; 446(7137):801-5. DOI: 10.1038/nature05654
Source: PubMed
ABSTRACT
Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown (reviewed in refs 1-3). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.

Full-text

Available from: Matthew Evans, Feb 12, 2015
LETTERS
Claudin-1 is a hepatitis C virus co-receptor required
for a late step in entry
Matthew J. Evans
1
*, Thomas von Hahn
1
*, Donna M. Tscherne
1
, Andrew J. Syder
1
, Maryline Panis
1
, Benno Wo
¨
lk
1
,
Theodora Hatziioannou
2
, Jane A. McKeating
1
{, Paul D. Bieniasz
2
& Charles M. Rice
1
Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver
cancer worldwide. A better understanding of the viral life cycle,
including the mechanisms of entry into host cells, is needed to
identify novel therapeutic targets. Although HCV entry requires
the CD81 co-receptor, and other host molecules have been impli-
cated, at least one factor critical to this process remains unknown
(reviewed in refs 1–3). Using an iterative expression cloning
approach we identified claudin-1 (CLDN1), a tight junction com-
ponent that is highly expressed in the liver
4
, as essential for HCV
entry. CLDN1 is required for HCV infection of human hepatoma
cell lines and is the first factor to confer susceptibility to HCV
when ectopically expressed in non-hepatic cells. Discrete residues
within the first extracellular loop (EL1) of CLDN1, but not protein
interaction motifs in intracellular domains, are critical for HCV
entry. Moreover, antibodies directed against an epitope inserted in
the CLDN1 EL1 block HCV infection. The kinetics of this inhibi-
tion indicate that CLDN1 acts late in the entry process, after virus
binding and interaction with the HCV co-receptor CD81. With
CLDN1 we have identified a novel key factor for HCV entry and
a new target for antiviral drug development.
Virus entry often requires sequential interactions between viral
proteins and multiple cellular factors. HCV virus particles are
believed to consist of a nucleocapsid surrounded by a lipid bilayer
studded with complexes of two envelope glycoproteins (HCVgp), E1
and E2. HCV entry is clathrin-dependent, requires a low pH com-
partment and occurs in a temperature-dependent manner
5–10
. Strong
evidence exists for the involvement of glycosaminoglycans and two
E2-binding proteins—scavenger receptor class B member I (SR-BI)
and the tetraspanin CD81—in HCV entry (reviewed in refs 1–3).
However, these molecules are insufficient for productive viral entry
because some cell lines express all three factors but do not support
HCV entry.
We performed a cyclic lentivirus based repackaging screen of a com-
plementary DNA library, derived from the highly HCV-permissive
hepatocarcinoma Huh-7.5 cell line
11
, for genes that render the non-
permissive CD81
1
SR-BI
1
293T cell line infectable with HIV-1 part-
icles pseudotyped with HCVgp (HCVpp) (Supplementary Fig. 1a and
Supplementary Methods). As a result of this screen CLDN1 was iden-
tified as a potential HCV entry factor (Supplementary Fig. 1a, b).
CLDN1 is one of 24 known claudin family members that form the
backbone of tight junctions through homo- and heterotypic inter-
actions (for a review see ref. 12). It is most highly expressed in liver,
but is also found in several other epithelial tissues
4
.Expressionof
CLDN1 specifically enhanced 293T susceptibility to diverse genotypes
of HCVpp by more than 100-fold, as determined by measuring luci-
ferase activity encoded by these pseudoparticles (Supplementary Fig.
1c and Fig. 1a). CLDN1 did not affect 293T susceptibility to pseudo-
particles bearing either no envelope proteins (Env
2
pp) or the unre-
lated vesicular stomatitis virus G protein (VSV-Gpp), which serve as
negative and positive infection controls, respectively.
We next sought to determine if CLDN1 overexpression renders
293T cells susceptible to infection with authentic cell culture grown
HCV (HCVcc)
13
. Whereas naive 293T cells showed no NS5A
1
foci, a
marker of ongoing virus replication, numerous NS5A
1
foci were
detected in cells expressing CLDN1 (Fig. 1b). The effective titre of
HCVcc was approximately 1,000-fold lower on the CLDN1 expres-
sing 293T cells as compared with Huh-7.5 cells, probably reflecting
low permissiveness for initiation of HCV RNA replication (as prev-
iously demonstrated for 293 cells when entry is bypassed by RNA
transfection
11,14
) rather than a defect in entry as HCVpp infectivity is
similar for CLDN1-expressing 293T and Huh-7.5 cells (Fig. 1a).
To test whether CLDN1 expression correlates with permissive-
ness for HCV entry, we surveyed the HCVpp susceptibility (Fig. 2a)
and CLDN1 expression level (Fig. 2b) for human cell lines pre- and
post-transduction with CLDN1 expression vectors. The HCVpp-
susceptible Huh-7.5 and Hep3B human hepatoma cell lines ex-
pressed endogenous CLDN1 and overexpression of CLDN1 in these
cells did not enhance HCV entry. Two HCVpp-resistant non-hepatic
cell lines, 293T and SW13, did not express detectable levels of endo-
genous CLDN1, but became highly susceptible to HCVpp on CLDN1
expression. The CD81-negative hepatoma HepG2 line, previously
*These authors contributed equally to this work.
1
Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave, New York 10021, USA.
2
Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First
Avenue, New York 10016, USA. {Present address: Division of Immunity and Infection, Institute of Biomedical Resear ch, Medical School, University of Birmingham, Birmingham B15
2TT, UK.
ab
Naive
CLDN1
RLU
(fold change relative
to mock control)
Env
pp
VSV-Gpp H77
(1a)
Con1
(1b)
OH8
(1b)
HCJ6
(2a)
JFH-1
(2a)
HCVpp
10
4
10
3
10
2
10
1
10
0
10
–1
Figure 1
|
CLDN1 expression confers susceptibility to HCV infection.
a
, Luciferase-encoding pseudoparticles bearing the indicated gps were used
to infect naive (white) or CLDN1-expressing (grey) 293T cells, or Huh-7.5
cells (black). For HCVpp the isolate name is given and the respective
genotype is indicated in parentheses. Values are normalized to the RLU
background measured in mock-infected cells (mean of n 5 3; error bars,
s.d.). RLU, relative light units.
b, Either naive or CLDN1-expressing 293T
cells were infected with HCVcc (J6/JFH chimaeric genome). At 72 h post
infection, cells were fixed with methanol and stained for NS5A antigen.
Representative visual fields are shown.
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reported to become susceptible on CD81 expression
6,15–17
, also
expressed endogenous CLDN1 and HCVpp entry remained CD81-
dependent even when CLDN1 was overexpressed. However, the
human HeLa and HepH cell lines (both CD81
1
SR-BI
1
CLDN1
2
)
remained HCVpp-resistant when overexpressing CLDN1 (Supplem-
entary Fig. 6a). Likewise, expression of human CLDN1 and/or CD81
in non-human cell lines did not allow HCVpp entry (Supplementary
Fig. 6b and data not shown). Moreover, murine CLDN1 (90% amino
acid identity to human) efficiently supported HCV entry, indicating
that CLDN1 is not a determinant of species host range (Supplemen-
tary Fig. 7). These observations suggest that one or more human-
specific HCV entry factor(s) remain to be discovered.
To determine if endogenous CLDN1 is required for HCV infection
of permissive liver cells, Hep3B cells were transfected with short
interfering RNAs (siRNAs) targeting CD81 or CLDN1. Expression
of the respective proteins was determined by flow cytometry (Fig. 3a).
Susceptibility to luciferase-encoding HCVpp (Fig. 3b), but not VSV-
Gpp (Fig. 3b) was markedly reduced in CLDN1-silenced cells com-
pared with cells treated with an irrelevant siRNA. When using six
distinct siRNAs of variable potency against CLDN1, HCVpp infec-
tivity was inversely correlated with the efficiency of silencing (Fig. 3c).
Furthermore, CLDN1 knockdown in Huh-7.5 cells potently inhib-
ited HCVcc infection (Fig. 3d, e). Given that even moderate down-
regulation of CLDN1 expression inhibited HCV infection, it is
conceivable that the relatively low-level CLDN1 expression in extra-
hepatic tissues
4
may be insufficient to support HCV entry and thus
CLDN1 may contribute to HCV tissue tropism.
Of the other claudin family members, neither CLDN7, the closest
relative of CLDN1 (60% amino acid identity), nor CLDN3, the clos-
est liver-expressed relative (49% identity), rendered 293T cells per-
missive to HCVpp (Fig. 4a), despite high levels of protein expression
(Fig. 4b). To map CLDN1-specific entry determinants, we analysed a
series of CLDN1–CLDN7 chimaeras (Fig. 4d) fused to the carboxy-
terminus of GFP. The parental fusion proteins functioned identically
to untagged proteins (Fig. 4e) and were expressed similarly (Fig. 4f).
The expression and membrane-localization of all mutants were con-
firmed by flow cytometry and confocal microscopy, respectively
(data not shown). CLDN1 is a 211 amino acid protein with four
transmembrane helices, intracellular amino and carboxy termini
and two extracellular loops (Fig. 4c)
4
. When extracellular loops were
exchanged between CLDN1 and CLDN7 (Fig. 4d) it was found that
only those containing the CLDN1 EL1 enhanced HCVgp-dependent
infection (Fig. 4e). Progressively smaller exchanges identified the
N-terminal third of the CLDN1 EL1 (EL1 N1/3) to be sufficient in
an otherwise CLDN7 background to confer full susceptibility to
HCVpp entry in 293T cells (Fig. 4e). The importance of CLDN1
EL1 in HCVpp entry was confirmed by deletion and insertional
mutagenesis (Supplementary Fig. 2a–c).
Five residues differ between CLDN1 and CLDN7 in the critical
region of EL1 (Fig. 4g). Although changing three of these in
CLDN7 to the corresponding CLDN1 sequence had no effect, intro-
duction of M32I or K48E into CLDN7 rendered 293T cells partially
HCVpp permissive (46% and 14% of wild-type CLDN1, respectively;
50
40
30
20
10
0
GFP-positive cells (%)
β-actin
Endog. CD81
p
TRIP-CLDN1
pTRIP-CD81
CLDN1
26
19
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Huh-7.5 SW13Hep3B HepG2
293T
64
49
a
b
Figure 2
|
CLDN1 expression is associated with susceptibility to HCVpp in
human cell lines. a
, The indicated human cell lines were either mock
transduced or transduced to express CLDN1 (‘pTRIP-CLDN1’), CD81
(‘pTRIP-CD81’), or both and then challenged with HCVpp (black bars) and
VSV-Gpp (white bars) encoding a GFP reporter. Background readings, that
is, percentage of GFP-positive cells seen with Env
2
pp, were subtracted for
each population (mean of n 5 3; error bars, s.d.).
b, CLDN1 expression was
assessed by immunoblotting. Approximate molecular weight (kDa) marker
positions are indicated to the left of each blot. Cell surface CD81 expression
(‘endog. CD81’) was determined before transduction, by flow cytometry
using anti-CD81 1.3.3.22 as a primary antibody.
CD81
CLDN1 (525)
CLDN1 (804)
CLDN1 (921)
CLDN1CD81
Hep3B cell staining
siRNA
CD81
CLDN1 (804)
CLDN1 (921)
siRNA
CLDN1CD81
Huh-7.5 cell staining
120
100
80
60
40
20
0
0
20
40
60
80
921
804
525
1259
306
635
Irr.
Relative
infectivity (%)
Reduction in CLDN1-
specific stain (%)
r = –0.86
P = 0.02
Irr.
CD81
525
804
921
CLDN1
150
125
100
75
50
25
0
Normalized RLU
Irr.
CD81
804
921
CLDN1
125
100
75
50
25
0
Normalized RLU
a
bc
de
Figure 3
|
CLDN1 silencing inhibits HCV entry. a, Dashed and solid lines
represent Hep3B cells treated with specific and an irrelevant siRNA,
respectively, and stained for CLDN1 (upper panels) or CD81 (lower panels).
The shaded area represents isotype-control-staining of specific siRNA-
treated cells.
b, HCVpp (black bars) and VSV-Gpp (white bars) infection of
siRNA-treated Hep3B cells normalized to irrelevant-siRNA-treated cells
(irr) (mean of n 5 3; error bars, s.d.).
c, Correlation between CLDN1
downregulation and HCV infectivity using six distinct siRNAs against
CLDN1. The x axis measures the reduction in D median fluorescence
intensity (CLDN1 stain minus isotype control stain) relative to irrelevant-
siRNA-treated cells; y-axis, HCVpp infectivity normalized to VSV-Gpp
infectivity. P, probability for no correlation as determined by Spearman’s
non-parametric test.
d, Silencing of CD81 and CLDN1 in Huh-7.5 cells.
e, siRNA-treated Huh-7.5 cells were infected with HCVcc encoding a
luciferase reporter (mean of n 5 4; error bars, s.d.).
LETTERS NATURE
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Fig. 4h) and the combination of both changes supported HCV entry
as efficient as wild-type CLDN1. Conversely, introduction of I32M or
E48K into CLDN1 dramatically reduced HCVpp susceptibility, and
the presence of both mutations reduced entry to background levels
(Fig. 4h). None of the wild-type or mutant claudin molecules affected
VSV-Gpp entry (Fig. 4h).
These data suggest that any CLDN1 features required for HCV
infection other than I32 and E48 must be conserved between
CLDN1 and CLDN7. In additional analyses, we found the CLDN1
C-terminal intracellular tail, which mediates interactions with other
TJ proteins
18
, and putative palmitoylation sites, which may regulate
protein interactions or direct the molecule to specific regions of
the plasma membrane
12
, were not required for HCVpp entry (Sup-
plementary Fig. 3a, b). This suggests a direct involvement of CLDN1
in HCV entry through an extracellular interaction rather than an
indirect effect mediated through intracellular interactions with other
tight junction components. All claudin mutants were also tested in
SW13 cells with similar results (data not shown), confirming that the
observed phenotypes were not 293T cell specific.
Blocking antibodies, ligands or antagonists are useful for probing
the function of cellular molecules involved in viral entry. Unfor-
tunately, all available CLDN1-specific antibodies recognize the intra-
cellular C-terminal segment of the protein and are thus not useful for
such studies. After an unsuccessful attempt to raise antibodies against
CLDN1 EL1 (data not shown), we identified a site in the C-terminal
portion of CLDN1 EL1 where a triple Flag epitope sequence could be
inserted without seriously impairing HCV entry (Supplementary Fig.
2a, c). HCVpp infection of 293T cells expressing this mutant (CLDN1
F10x3) was blocked in a dose-dependent manner by anti-Flag M2
monoclonal antibody. As controls, neither HCVpp infection of cells
expressing wild-type CLDN1 nor VSV-Gpp infection of cells expres-
sing CLDN1 F10x3 were affected by equal amounts of antibody
(Fig. 5a and Supplementary Fig. 4). Although these results further
suggest that CLDN1 functions in HCV entry through a direct inter-
action between EL1 and the virion, evidence for direct HCV binding
to CLDN1 is lacking. In fact, HCVcc binding to CHO cells was only
enhanced by expression of human SR-BI, but not CLDN1 or CD81
(Supplementary Fig. 8). However, virus–receptor interactions before
CLDN1 engagement may trigger HCV glycoprotein conformational
changes required for CLDN1 binding, paralleling the situation with
HIV-1 and its co-receptor CCR-5, in which binding requires prior
interaction with CD4 (ref. 19).
To determine when CLDN1 functions in HCV entry, we examined
the ability of the M2 antibody to block infection of CLDN1 F10x3
cells when added at various times during cell entry. To synchronize
infection, 293T cells expressing CLDN1 F10x3 were incubated with
HCVpp for 2 h at 4 uC, so that virion binding but not entry could
occur, and then washed and shifted to 37 uC, allowing the infection to
continue (see Supplementary Methods). Antibodies directed against
both CD81 (JS81) and the Flag epitope (M2) inhibited HCVpp infec-
tion and retained maximal inhibitory activity even after the temper-
ature shift to 37 u C, indicating that like CD81 (ref. 10), CLDN1 acts at
the post-binding stage of HCV entry (Fig. 5b). However, the inhib-
itory activity of anti-CD81 was lost much earlier than that of anti-
Flag (half-maximal inhibition at 18 and 73 min post temperature
shift, respectively), suggesting a sequence of events in which CD81
acts prior to CLDN1 in HCV entry.
To test if CLDN1 is required for HCVgp-mediated membrane
fusion we used a cell–cell fusion assay where 293T ‘acceptor’ cells,
encoding a HIV-1 transactivator of transcription (Tat)-dependent
GFP, were co-cultured with Tat-expressing ‘donor’ cells such that
fusion between donor and acceptor cell membranes results in GFP ex-
pression (Supplementary Fig. 5 and Supplementary Methods). When
using donor cells expressing HCV E1E2, expression of CLDN1 on the
acceptor cells resulted in a sevenfold increase in the number of GFP-
positive foci per well (P , 0.001 by unpaired t-test), indicating a sig-
nificant enhancement of HCVgp-dependent cell–cell fusion (Fig. 5c).
A pH 5 wash did not significantly increase the number of GFP foci
observed, but changed their appearance from clusters of discrete GFP-
positive cells to large brightly GFP-positive syncytia, indicating that the
pH-dependence of HCVgp-mediated fusion
5–10
is maintained in this
assay (Supplementary Fig. 5). These results demonstrate that CLDN1
is required for HCVgp-dependent cell fusion although it is presently
unclear whether the protein participates directly in the fusion process
or acts at an earlier step that is required to enable subsequent fusion.
The requirement for numerous cellular factors including glycos-
aminoglycans, SR-BI, CD81 and now CLDN1, indicates that HCV
cell entry is a complex multi-step process (reviewed in refs 1–3). An
30
20
10
0
GFP-positive cells (%)
cab
Extracellular
N
C
EL1
EL2
26
19
64
49
26
19
26
19
CLDN1CLDN3CLDN7
β-actin
Mock
CLDN1
CLDN3
CLDN7
Mock
CLDN1
CLDN3
CLDN7
Normalized RLU
GFP–CLDN7 w/ CLDN1
GFP–CLDN1
w/ CLDN7
EL1 EL2 EL1 EL2 EL1
N1/2
EL1
C1/2
e
Mock
GFP
GFP–
CLDN1
GFP–
CLDN7
EL1
N1/3
Normalized RLU
Q31R M32I S33Y I41V K48E M32I
K48E
I32M E48K I32M
E48K
d
g
h
GFP–CLDN1 w
/
GFP–CLDN7 w/
10
1
10
0
10
3
10
2
10
–1
10
–2
10
1
10
0
10
3
10
2
10
-1
10
-2
GFP–
CLDNs
GFP
β-actin
60
50
40
25
60
50
GFP–CLDN1
GFP–CLDN7
GFP
Mock
f
EL1 N1/3
CLDN1
CLDN7
P
.
Q
.
W
.
S
.
Y
.
A
.
G
.
D
.
N
.
I
.
T
.
A
.
Q
.
A
.
M
.
Y
.
G
.
R
Q
31
M
I
32
Y
S
33
E
K
48
V
I
41
Figure 4
|
HCVpp susceptibility depends on residues in the first
extracellular loop of CLDN1. a, GFP-reporter HCVpp (black) and VSV-Gpp
(white) infection of 293T cells expressing CLDN1, 3 or 7. Background
readings, that is, percentage of GFP-positive cells seen with Env
2
pp, were
subtracted for each population (mean of n 5 3, error bars, s.d.).
b, Immunoblot for CLDN1, 3 and 7. Approximate molecular weight (kDa)
marker positions are indicated to the left of each blot.
c, CLDN1 topology.
d, CLDN1/CLDN7 chimaeras with regions of CLDN1 and CLDN7
represented as dark and light lines, respectively. All chimaeras were
N-terminally GFP-tagged.
e, HCVpp (black) and VSV-Gpp (white) infection
of 293T cells expressing the chimaeras depicted above in panel
d. For
chimeras, the x axis labels refer to the GFP–CLDN1 or GFP–CLDN7 fusion
backbone encoding (‘w/’) the indicated region of the other claudin protein.
N and C indicate either the amino or carboxy terminal, respectively, piece of
CLDN1 EL1, swapped into CLDN7. Readings are normalized to 293T cells
expressing wild-type CLDN1 (mean of n 5 4; error bars, s.d.).
f, Immunoblot
for GFP.
g, Alignment of the N-terminal half of EL1 in CLDN1 and CLDN7.
Identical sequences are represented by a full-stop, numbering represents
amino acid position in full-length CLDN1.
h, 293T cells expressing the
indicated point mutants were tested for susceptibility as described above
(mean of n 5 4; error bars, s.d.).
NATURE
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important challenge is to delineate the precise function that each
cellular entry factor fulfils. Our data suggest a role for CLDN1 late
in the HCV entry process, perhaps downstream of interactions with
other virus-receptor/co-receptors such as CD81. However, differ-
ences in blocking antibody efficacy are an important caveat to bear
in mind when interpreting the inhibition data in Fig. 5b.
Preliminary studies indicate that CLDN1 is localized on the plasma
membrane in HCV permissive cells, is enriched at sites of cell–cell
contact and co-localizes with a sub-fraction of CD81 (unpublished
data). Interestingly, an association of tetraspanins with claudin-11
has been reported
20
. It should be emphasized, however, that the
situation may differ in polarized hepatocytes, where CLDN1 is
strictly localized to tight junction regions
21
. This location fits well
with a co-receptor rather than a primary receptor role for CLDN1.
Other tight-junction-associated molecules have been found to serve
as (co-) receptors for reoviruses
22
, coxsackievirus B and certain ade-
noviruses
23
. Coxsackievirus B entry requires initial binding to a prim-
ary receptor (decay-accelerating factor) on the luminal cell surface,
followed by lateral migration of the virus–receptor complex to the
tight junction, where interaction with the CAR co-receptor and
uptake into the host cell occur
24
. A similar pathway is conceivable
for HCV, and it will be of considerable interest to devise systems to
examine HCV entry in polarized cells.
The involvement of CLDN1 in HCV entry raises interesting
questions regarding the pathogenesis of hepatitis C. Loss of cell–cell
contacts is a hallmark of malignant transformation and altered
expression of CLDN1 and other claudin family members has been
reported in a number of human malignancies including hepatocel-
lular carcinoma—the most lethal complication of chronic HCV
infection
25–27
. In addition, the extremely high viral loads observed
in some immunosuppressed liver transplant recipients could con-
ceivably compromise tight junction integrity and contribute to the
development of cholestatic hepatitis
28,29
. Finally, CLDN1 presents a
new target for HCV therapeutic intervention that may complement
ongoing efforts to block intracellular replication events with inhibi-
tors of the HCV proteases and polymerase.
METHODS
For more detailed methods see Supplementary Information.
Pseudoparticles. All pseudoparticles were generated by 293T FuGENE6 (Roche
Applied Science, Indianapolis) cotransfection of plasmids encoding (1) a min-
imal HIV provirus (such as pTRIP, V1 or CSxW) encoding a reporter gene or
other transgene, (2) HIV gag-pol and (3) an appropriate viral gps (HCV E1E2 or
VSV-G). The pNL43.luc.R
2
.E
2
provirus encodes gag-pol and only requires
the addition of the gps
5
. To generate Env
2
pp the glycoprotein vector was
replaced with empty vector. Pseudoparticle-containing supernatants were har-
vested at 48 and 72 h were pooled and filtered (0.45 mm mesh). Pseudoparticle
infections were performed in the presence of 4 mgml
21
polybrene. A minimum
of 48 h elapsed between transduction and reporter gene quantification or sub-
sequent experiments.
Cyclic lentivirus cDNA library repackaging screen. The V1 lentiviral Huh-7.5-
derived cDNA library was packaged into VSV-Gpp and applied to 293T cells,
which were then challenged with HCVpp, encoding either a puromycin (CSPW)
or zeocin (CSZW) reporter gene, and subjected to antibiotic selection (Sup-
plementary Fig. 1a). Surviving clones were pooled and a fraction tested for their
susceptibility to GFP-encoding HCVpp. The populations were subsequently
either challenged with a different antibiotic resistance gene for further selection
or transfected with HIV gag-pol and VSV-G expression plasmids to package the
V1 genomes with the cDNA inserts contained in the surviving 293T cells into
fresh VSV-Gpp (cyclic packaging rescue, CPR). These were used to transduce
naive 293T cells for subsequent rounds of screening. Deletions in the 39-long
terminal repeat of the CSPW and CSZW proviruses prevented their re-packaging
such that the newly transduced 293T cells could again be challenged as outlined
above.
Cell culture grown HCV (HCVcc). Plasmids encoding the chimaeric J6/JFH
genome
13
and FL-J6/JFH-59C19Rluc2AUbi reporter genome
9
were XbaI linear-
ized and transcribed using MEGAscript T7 (Ambion, Austin, Texas). RNA was
electroporated into Huh-7.5 cells using a ECM 830 electroporator (BTX
Genetronics). High-titre stocks were generated by serial passage through naive
Huh-7.5 cells.
Received 18 December 2006; accepted 6 February 2007.
Published online 25 February; corrected 12 April 2007 (details online).
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aCD81
aFLAG
None
IgG
JS81 anti-CD81
M2 anti-Flag
RLU (% no antibody)
120
100
80
60
40
20
0
100
0
1
0.1
10
Wild type F10x3
120
100
80
60
40
20
0
–180
–120
0
120
180
240
Time (min)
GFP foci per well
40
20
0
30
50
10
ab c
g ml
–1
)
Antibody
100
0
100
0
100
0
+Tat
+E1E2
+Tat
+E1E2
Donor cell
Naive
60
Per cent entry (%)
Figure 5
|
Analysis of CLDN1 function in the HCV entry process. a, 293T
cells expressing wild-type or F10x3 CLDN1 were challenged with HCVpp
(black) or VSV-Gpp (white) in the presence or absence of M2 anti-Flag.
(Values were normalized to RLU from cells with no antibody; mean of n 5 4;
error bars, s.d.).
b, Synchronized infections were performed on 293T cells
expressing F10x3 CLDN1, with the indicated antibodies being present from
the time indicated on the x axis. Values (per cent entry) are relative to the
signal seen when antibody was added 4 h post temperature shift. Controls
are normalized to the value for M2 added at 4 h. Fits of t 5 0 and later data
points represent a one-phase exponential association and sigmoidal
dose–response (variable slope) for JS81 and M2, respectively (mean of n 5 3;
error bars, s.d.).
c, Cell fusion assay using 293T acceptor cells expressing Tat-
regulated GFP alone (white) or together with CLDN1 (black), in co-culture
with 293T donor cells expressing no transgene (naive), HIV-1 Tat, HCV
E1E2 or both (mean of n 5 3; error bars, s.d.).
LETTERS NATURE
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements The authors thank J. Tassello, M. Hunter and N. Torres for
excellent technical assistance; S. You for providing HCVcc/Rluc virus stocks;
D. Schmid and M. Landthaler for expert advice on RNAi; and M. MacDonald and
L. Dustin for reviewing the manuscript. This work was supported by the Greenberg
Medical Research Institute, the Ellison Medical Foundation, the Starr Foundation,
the Ronald A. Shellow Memorial Fund, the Richard Salomon Family Foundation, and
the National Institutes of Health (grants to M.J.E., T.v.H., D.M.T., A.J.S., P.D.B. and
C.M.R.). P.D.B. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist. C.M.R. is
an Ellison Medical Foundation Senior Scholar in Global Infectious Diseases. T.v.H.
and B.W. were supported by postdoctoral fellowships from the Deutsche
Forschungsgemeinschaft. This work was presented in part at the 13th International
Meeting on Hepatitis C Virus & Related Viruses, Cairns, Australia, 27
31 August,
2006.
Author Contributions M.E., T.v.H. and C.M.R. designed the project, analysed
results and wrote the manuscript. M.E., T.v.H., D.M.T., A.J.S., M.P. and B.W.
performed the experimental work. T.H. and P.D.B developed the screening
technology and assisted in its implementation. T.v.H. and J.A.M were involved in
preliminary experiments identifying and characterizing HCV nonpermissive cell
lines.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financi al interests:
details accompany the full-text HTML version of the paper on www.nature.com/
nature. Correspondence and requests for materials should be addressed to C.M.R.
(ricec@mail.rockefeller.edu).
NATURE
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  • Source
    • "Moreover, the receptors are regarded as promising targets for development of novel antivirals [44][45][46][47]. While reporter-expressing pseudoparticles are widely used to screen viral receptors [48,49], RCREVs carrying Fluc [18,50], GFP [51] or Neo R [52] as new useful tools have been applied for screening of viral receptors (Table 1). Since RCREVs can infect the cells with multiple life cycles in contrast to pseudoparticles, more false-positive receptors may be screened. "
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  • Source
    • "Scavenger receptor class B type 1 (SR-B1) was also identified as a co-receptor responsible for E2 binding to human hepatic cells by comparative binding studies [5]. Upon introduction of pseudotype particles bearing HCV envelope proteins (HCVpp) [6], claudin-1 (CLDN1) and occludin (OCLN) were identified as entry receptors for HCVpp into human kidney-derived HEK293 cells and mouse embryonic fibroblast-derived NIH3T3 cells, respectively [7, 8]. CD81, SR-B1, CLDN1 and OCLN are regarded as essential factors for HCV entry because mouse NIH3T3 cells and hamster CHO cells expressing these four factors permit entry of HCVpp [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV.
    Full-text · Article · May 2016 · PLoS Pathogens
  • Source
    • "Structural analysis of E2 (Khan et al., 2014; Kong et al., 2013) has revealed antibody and receptor binding sites. Virus entry into hepatocytes occurs as a complex cascade requiring the cellular factors CD81 (Zhang et al., 2004), scavenger receptor BI (SR-B1, Bartosch et al., 2003c) and two tight junction proteins claudin-1 (Evans et al., 2007) and occludin (Ploss et al., 2009). EGFR and EphA2 also contribute to entry (Lupberger et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization.
    Full-text · Article · Mar 2016 · Virology
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