JOURNAL OF VIROLOGY, Apr. 2008, p. 3466–3479
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 7
Scavenger Receptor Class B Is Required for Hepatitis C Virus Uptake
and Cross-Presentation by Human Dendritic Cells?
Heidi Barth,1,2* Eva K. Schnober,1,3,4Christoph Neumann-Haefelin,1Christine Thumann,3,5
Mirjam B. Zeisel,3,5Helmut M. Diepolder,6Zongyi Hu,2T. Jake Liang,2Hubert E. Blum,1
Robert Thimme,1Me ´lanie Lambotin,3,5and Thomas F. Baumert1,3,5,7*
Department of Medicine II, University of Freiburg, Freiburg, Germany1; Liver Diseases Branch, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland2; Inserm, U748, Strasbourg, France3;
Faculty of Biology, University of Freiburg, Freiburg, Germany4; Universite ´ Louis Pasteur, Strasbourg, France5;
Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany6; and
Service d’He ´patogastroente ´rologie, Centre Hospitalier Universitaire Strasbourg, Strasbourg, France7
Received 17 November 2007/Accepted 11 January 2008
Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most
recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion
and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host
factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs).
Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels,
human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human
hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle bind-
ing, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically
modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function
of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived
HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake
and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic
approaches aiming at the induction of efficient antiviral immune responses.
Scavenger receptor class B type I (SR-BI) and its splicing
variant SR-BII are human high-density lipoprotein (HDL) re-
ceptors with an identical extracellular domain. These receptors
mediate HDL binding, followed by selective uptake of choles-
terol and cholesteryl ester in the liver and steroidogenic tissues
(16). Recently, SR-BI and SR-BII have been found to mediate
the binding and uptake of a broad range of bacteria into
nonphagocytic human epithelial cells overexpressing SR-BI
and SR-BII (50, 60), suggesting that SR-Bs may serve as pat-
tern recognition receptors for bacteria. Furthermore, most re-
cent studies have indicated that SR-BI is an important host
entry factor for hepatitis C virus (HCV) infection of hepato-
cytes (25, 31, 69).
HCV is a noncytopathic, hepatotropic member of the Fla-
viviridae family that causes chronic hepatitis, liver cirrhosis,
and hepatocellular carcinoma (13). Resolution of HCV infec-
tion is associated with a vigorous, long-lasting, HCV-specific
CD4?(helper) and CD8?(cytotoxic) T-cell response (9, 57),
whereas such responses are usually weak or absent in chronic
hepatitis C. The priming and expansion of naı ¨ve T cells depend
on efficient antigen presentation and stimulation by dendritic
cells (DCs), which among several unique features have the
ability to crossover exogenous antigens to the endogenous
pathway to gain access to major histocompatibility complex
(MHC) class I-inducing CD8?T-cell responses. This process,
called cross-presentation, results in cytotoxicity against viruses
that have restricted tissue tropism (1). DCs express numerous
receptors involved in the recognition and endocytosis of a large
number of pathogens, as well as self antigens (23) such as
Fc?-receptors, Toll-like receptors, C-type lectins, and SRs (45,
52). The presence of both positive-strand HCV RNA and its
replicative intermediates (negative-strand HCV RNA) in DCs
from patients infected with HCV suggests that DCs may be
permissive for HCV infection (24, 33, 48). However, the viral
load detected in DCs from patients infected with HCV is
extremely low compared to the viral load in infected hepato-
HCV-like particles (HCV-LPs) generated by self-assembly
of the HCV structural proteins core, E1, and E2 in insect cells
exhibit antigenic properties similar to those of virions isolated
from HCV-infected patients (7) and recombinant infectious
virions synthesized in tissue culture (cell culture-derived HCV
[HCVcc]) (38, 63, 70). Recently, we have shown that HCV-LPs
are efficiently taken up by human monocyte-derived DCs and
defined subsets of blood DCs in an envelope- and receptor-
mediated manner (5). Following HCV-LP uptake, DCs effi-
ciently activate HCV-specific CD8?T cells (5), indicating
MHC class I presentation of HCV-LP-derived peptides in the
absence of viral replication. Thus, HCV-LPs represent a
* Corresponding author. Mailing address for Heidi Barth: Liver
Diseases Branch, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, 10 Center Drive,
Bethesda, MD 20892. Phone: (301) 402-5113. Fax: (301) 402-0491.
E-mail: email@example.com. Mailing address for Thomas F. Baumert:
Inserm Unit 748, Service d’He ´patogastroente ´rologie, Universite ´ Louis
Pasteur, 3 Rue Koeberle ´, F-67000 Strasbourg, France. Phone: 33-3 90
24 37 02. Fax: 33-3 90 24 37 23. E-mail: Thomas.Baumert@viro-ulp
?Published ahead of print on 23 January 2008.
unique model system to study the cellular and molecular mech-
anisms of HCV uptake and cross-presentation. The host entry
factors mediating the uptake and cross-presentation of HCV-
LPs into DCs are unknown. The identification of these factors
would not only help in understanding the molecular mecha-
nism of HCV entry and presentation but also guide the devel-
opment of therapeutic interventions to modulate the HCV-
specific T-cell response.
In this study, we demonstrate that SR-BI plays a crucial role
in mediating the first steps of HCV-LP–DC interaction and
represents a cell surface receptor for HCV entry into DCs. The
involvement of SR-BI in HCV-LP-mediated cross-presenta-
tion suggests a functional role for SR-BI in the initiation of
HCV-specific immune responses.
MATERIALS AND METHODS
Recombinant proteins, antibodies, and cells. HCV-LPs were synthesized in
Sf9 insect cells as previously described (65). The HCV-LP E2 concentration was
determined as previously described (15). Mouse anti-E2 monoclonal antibodies
(MAb) (16A6 and AP33), mouse anti-core MAb (C1 and C2), mouse anti-E1
(1C4), and chimpanzee anti-E2 MAb (49F3) have been previously described (5,
65). Polyclonal antibodies against the extracellular loop of SR-BI were raised by
genetic immunization of BALB/c mice or Wistar rats with a plasmid expressing
the full-length human SR-BI cDNA. The SR-BI plasmid (pcDNA CLA-1) was
kindly provided by T. Huby (Inserm, Dyslipoproteinemia and Atherosclerosis
Research Unit, Ho ˆpital de la Pitie ´, Paris, France) (40). Preimmune serum was
collected from mice and rats before immunization. Immunoglobulin G (IgG)
from preimmune and anti-SR-BI-positive sera were purified by using a MabTrap
kit (Amersham Biosciences, Freiburg, Germany) according to the manufactur-
er’s instructions, as described previously (69). Anti-human CD36 (FA6-152)
MAb was obtained from Immunotech (Marseille, France), anti-human LOX-1
(23C11) MAb from HyCult Biotechnology (Uden, The Netherlands), anti-hu-
man CD81 (JS81) from BD Pharmingen (San Jose, CA), and rabbit anti-SR-BI
(NB 400-104) and anti-SR-BII (NB 400-102) polyclonal sera from Novus Bio-
logicals (Littleton, CO). Fucoidan, poly(C), lactacystin, and lipopolysaccharide
(LPS; Escherichia coli 026:B6) were obtained from Sigma-Aldrich (St. Louis,
MO). HDL and low-density lipoprotein (LDL) were isolated from plasma of
healthy individuals by ultracentrifugation and dialyzed against phosphate-buff-
ered saline (PBS). Lipoprotein cholesterol concentrations were determined as
described previously (47). Oxidized LDL was generously provided by O. Que-
henberger (Department of Medicine, University of California, San Diego, CA).
The isolation of LDL from human plasma and oxidation of LDL were performed
as previously described (62). The origins and maintenance of HepG2, Sf9 insect,
and CHO cells have been described previously (54, 65). CD19?and CD4?cells
were isolated using anti-CD19 and anti-CD4 MAb-coated magnetic beads fol-
lowing the manufacturer’s instructions (Miltenyi Biotech, Bergish Gladbach,
Germany). DCs were generated from peripheral blood mononuclear cells of
healthy, anti-HCV-negative blood donors (for DC activation, particle binding,
and uptake studies), as well as an individual with chronic HCV infection (for the
study of HCV-LP cross-presentation), as described previously (5). In brief,
CD14?cells were purified from peripheral blood mononuclear cells by using
CD14 MicroBeads (Miltenyi Biotech) according to the manufacturer’s instruc-
tions. To obtain monocyte-derived DCs, CD14?cells were cultured in the pres-
ence of interleukin-4 (1,000 U/ml) and granulocyte-macrophage colony-stimu-
lating factor (800 U/ml) (CellGenix, Freiburg, Germany). Immature DCs were
collected on day 5 and assessed by cytofluorimetric cell surface phenotyping
using anti-human CD80-, CD83-, CD86-, and HLA-DR–phycoerythrin (PE)-
conjugated antibodies. To study whether IgG purified from anti-SR-BI or control
serum induces DC maturation, immature DCs were exposed to anti-SR-BI IgG
or control IgG (50 ?g/ml) for 16 h. DC maturation was analyzed by flow cytom-
etry of CD80, CD83, CD86, and HLA-DR surface expression.
SR expression. For the analysis of SR expression, cells (1 ? 105cells/100 ?l)
were incubated with antibodies directed against SR-BI, LOX-1, CD36, antigal-
actosidase antibody (control IgG), or preimmune serum (control serum). Sub-
sequently, cells were incubated with PE-conjugated anti-mouse IgG and analyzed
by fluorescence-activated cell sorter (FACS) as recently described (5). For the
FACS analysis of SR-BI and SR-BII, cells were permeabilized with 0.05 to 0.1%
saponin prior to incubation with rabbit polyclonal anti-SR-BI/II serum and
allophycocyanin-conjugated anti-rabbit IgG. To demonstrate the specificity of
polyclonal anti-SR-BI serum, CHO cells were transfected with pcDNA3 (control
vector) or pcDNA-SR-BI by using liposome-mediated gene transfer (Lipo-
fectamine; Invitrogen, Karlsruhe, Germany) according to the manufacturer’s
instructions (2, 54). The CHO cells were then incubated with polyclonal anti-
SR-BI serum or preimmune serum and analyzed for SR-BI expression by flow
cytometry as described above.
HCV-LP binding and uptake. Cells (1 ? 105cells/100 ?l) were incubated with
HCV-LPs (HCV-LP E2 concentration of 1 ?g/ml) (5) corresponding to approx-
imately 5 ? 109virus particles/100 ?l or about 50,000 viral particles per cell
(according to Yu et al. ) or with insect cell control preparations (derived from
insect cells infected with a recombinant baculovirus containing the cDNA for
?-glucuronidase [GUS]) (65) for 1 h at 4°C, and cell-bound HCV-LPs were
detected by using mouse (AP33) or chimpanzee anti-E2 MAb (49F3) and FACS
as described previously (5). To assess the inhibition of HCV-LP binding by
antibodies directed against SRs or CD81, cells were preincubated with anti-
SR-BI (1:10 or 1:20 dilution), anti-CD36, anti-CD81, control IgG (50 ?g/ml
each), preimmune serum (1:10 or 1:20 dilution), or anti-SR-BI IgG and control
IgG purified from serum (100 ?g/ml each) in PBS for 1 h at 4°C. Then, HCV-LPs
were added for 1 h at 4°C. The cellular binding of HCV-LPs was quantified by
FACS using chimpanzee anti-E2 (49F3) or mouse anti-E2 (AP33) MAb and
PE-conjugated anti-human or anti-mouse IgG antibody. To study whether cel-
lular HCV-LP binding was affected by SR-B ligands, human serum, or lipopro-
teins, HCV-LPs were preincubated with fucoidan, poly(C), HDL, LDL, and
oxidized LDL at different concentrations for 1 h at room temperature. Then,
HCV-LP–ligand complexes were added to the cells for 1 h at 4°C and cell-bound
HCV-LPs were detected as described above. To analyze the uptake of HCV-LPs,
DCs were incubated with HCV-LPs or GUS for 3 h at 37°C, and internalized
particles were stained using mouse anticore antibody (C1 and C2), human
anti-E1 antibody (1C4), mouse anti-E2 (AP33), or chimpanzee anti-E2 antibody
(49F3). To assess the inhibition of HCV-LP uptake, HCV-LPs were preincu-
bated for 1 h at 37°C with mouse anti-E2 antibody (AP33) or mouse control IgG
(each 100 ?g/ml) in PBS. Then, HCV-LP–antibody complexes were added to
DCs and incubated for 3 h at 37°C. The uptake of HCV-LPs by DCs was
determined by immunofluorescence and confocal laser scanning microscopy
(LSM) as described previously (5).
HCV-LP cross-presentation. To study the role of SRs in HCV-LP cross-
presentation, we analyzed HCV-LP-mediated antigen cross-presentation using
HCV core-specific CD8?T cells. Peripheral HCV core-specific CD8?T cells
(recognizing an epitope in the HCV core protein comprising amino acids 36 to
53) were generated from a patient chronically infected with HCV, as described
previously (5). After preincubation of autologous DCs with anti-SR-BI serum,
preimmune serum (1:10 dilution), anti-CD81, control IgG (50 ?g/ml each), or
SR ligand fucoidan and poly(C) (1 ?g/ml each) for 1 h, HCV-LPs (corresponding
to an E2 concentration of 2.5 ?g/ml) or insect cell control preparations were
added to the DCs for 1 h at 37°C. Then, cells were extensively washed to remove
unbound HCV-LPs and cultured for 4 h at 37°C, allowing efficient uptake and
antigen processing. During the final 16 h, CD40 ligand (1 ?g/ml) was added to
the culture medium as a maturation stimulus. The DCs were then extensively
washed and cocultured with HCV core-specific CD8?T cells at a ratio of 1:2.
After 5 h of incubation, intracellular gamma interferon (IFN-?) staining of
core-specific CD8?T cells was performed as recently described (58). To study
the mechanisms of HCV-LP antigen processing, DCs were preincubated for 1 h
at 37°C in the absence or presence of increasing concentrations of lactacystin (0
to 50 ?M), a highly specific proteasome inhibitor. After preincubation, HCV-LPs
were added to the wells in the continuous presence of inhibitor for 4 h at 37°C
and cross-presentation was analyzed as described above. Approval of the studies
was obtained from the Freiburg University Hospital institutional review board.
Informed consent was obtained according to the Declaration of Helsinki.
Uptake of HCVcc into DCs. HCVcc were generated as previously reported
(63). To obtain high-titer and purified HCVcc, the culture medium of JFH1-
infected Huh7.5.1. cells was concentrated and subjected to iodixanol density
gradient ultracentrifugation. Then, the gradient fractions were collected and
analyzed for HCV RNA and infectivity titers as recently described (32). To study
HCVcc uptake into DCs, DCs (1 ? 105cells/100 ?l) were incubated with HCVcc
(5 ? 108to 1 ? 109copies/ml, corresponding to an infectivity titer of 1 ? 105
focus-forming units/ml) for 2 h at 4°C, followed by a temperature shift to 37°C for
2 h. Following the incubation at 37°C, the DCs were washed, fixed, and perme-
abilized. HCVcc uptake was detected by using mouse monoclonal anti-E2 anti-
body (AP33) and the protocol described above for HCV-LPs. For costaining of
cytoplasmic structures, cells were coincubated with an anti-human actin anti-
body. To assess the inhibition of HCVcc uptake by anti-SR-BI IgG, DCs were
preincubated for 1 h at 37°C with purified rat anti-SR-BI IgG or rat control IgG
(250 ?g/ml). Then, HCVcc were added as described above and the uptake of
VOL. 82, 2008SR-BI AND HCV CROSS-PRESENTATION 3467
HCVcc by DCs was determined by immunofluorescence and confocal LSM
analysis as described above. The HCVcc uptake was quantified by counting the
average number of cells with positive staining for HCV E2 protein per total cells
(n ? 300) in the presence or absence of anti-SR-BI IgG or control IgG.
High level of expression of SR-Bs on human DCs. SR-BI is
a type III transmembrane protein that crosses the membrane
twice to form a heavily glycosylated extracellular loop with two
short intracellular tails. SR-BI and its isoform SR-BII are iden-
tical except for the region encoding the C-terminal cytoplasmic
domain, suggesting that alternative splicing of a single tran-
script yields two distinct mRNAs (64). Using commercially
available rabbit polyclonal anti-SR-BI and SR-BII antibodies
directed against the C-terminal cytoplasmic tail of SR-BI and
SR-BII, we demonstrated that DCs are characterized by high
levels of expression of SR-BI and SR-BII on their cell surface
Since antibodies targeting the C-terminal cytoplasmic tail of
SR-BI or SR-BII do not interfere with SR-B–ligand interac-
tion, we generated a polyclonal anti-SR-BI antibody directed
against the extracellular loop of SR-BI by genetic immuniza-
tion with a plasmid carrying the full-length human SR-BI
cDNA. To demonstrate that the resulting anti-SR-BI antibod-
ies specifically interact with human SR-BI, we studied the
binding of anti-SR-BI to CHO cells expressing human SR-BI
on their cell surface. As shown in Fig. 1B, human SR-BI-
transfected CHO cells specifically interacted with anti-SR-
BI antibodies. By contrast, there was no interaction between
CHO cells transfected with control vector and anti-SR-BI se-
rum or CHO cells transfected with SR-BI cDNA and preim-
mune serum (Fig. 1B). In addition, polyclonal anti-SR-BI se-
FIG. 1. SR-BI and SR-BII expression on human DCs. (A) SR-BI and
SR-BII expression on human monocyte-derived DCs. Following fixation
and permeabilization, DCs were incubated with rabbit anti-SR-BI (NB
400-104) and anti-SR-BII (NB 400-102) polyclonal antibodies directed
against the SR-B cytoplasmic domain and subsequently stained with al-
lophycocyanin-conjugated goat anti-rabbit IgG. Cells stained with the
secondary antibody alone served as negative controls (gray-shaded
curves). The x and y axes show mean fluorescence intensities and relative
numbers of stained cells, respectively. (B) Specific binding of mouse anti-
human SR-BI to SR-BI expressed in CHO cells. Anti-SR-BI polyclonal
serum directed against the SR-BI extracellular loop was raised by genetic
immunization of BALB/c mice with a plasmid carrying the full-length
human SR-BI cDNA. CHO cells were transfected with pcDNA-SR-BI or
control vector (pcDNA). Flow cytometry of SR-BI-transfected CHO cells
incubated with mouse anti-human SR-BI polyclonal serum and PE-con-
jugated anti-mouse IgG demonstrated specific interaction of anti-SR-BI
antibodies with human SR-BI. Numbers inside the panels represent the
percentage of positively stained cells in relationship to the total number of
cells. (C) Detection of cell surface SR-BI on DCs by anti-SR-BI. DCs
were incubated with anti-SR-BI or preimmune serum and subsequently
stained with PE-conjugated anti-mouse IgG. Cells stained with the sec-
ondary antibody alone served as negative controls (gray-shaded curves).
CD, SR-BI/II cytoplasmic domain; EL, SR-BI extracellular loop; FL4/
2-H, fluorescence 4/2-height.
FIG. 2. SR expression on DCs and other cell types. Cell surface
expression of SR was determined by flow cytometry using antibodies
directed against SR-BI, CD36, LOX-1, or control antibody and pre-
immune serum. In addition, cells were stained for CD81 expression
using a monoclonal anti-human CD81 antibody. Histograms corre-
sponding to cell surface expression of the respective cell surface mol-
ecules (open curves) are overlaid with histograms of cells incubated
with the appropriate isotype control (gray-shaded curves [NC]).
FL2-H, fluorescence 2-height.
3468 BARTH ET AL.J. VIROL.
rum, but not preimmune serum, bound to the cell surface of
human monocyte-derived DCs, suggesting a specific binding to
SR-BI expressed on the DC surface (Fig. 1C). Taken together,
these findings demonstrate that polyclonal anti-SR-BI directed
against epitopes of the SR-BI extracellular loop specifically
recognizes human SR-BI expressed on the surface of DCs.
Next, we analyzed the expression profiles of other SRs on
the surface of DCs and other cell types. As shown in Fig. 2,
DCs expressed high levels of SR-BI, which were comparable to
the levels of SR-BI expressed on human HepG2 hepatoma
cells. By comparison, cell-surface SR-BI expression on mono-
cytes and T and B cells was weak or absent (Fig. 2). CD36,
another member of the class B family, was highly expressed
both on monocytes and on DCs and human HepG2 hepatoma
cells (Fig. 2). The expression of LOX-1, a member of the class
E family, was very weak or absent on DCs (Fig. 2).
SR-BI and HDL mediate binding of HCV-LPs to DCs. Re-
cent evidence suggests that the function of SR-BI and SR-BII
is not linked only to lipoprotein metabolism. SR-BI and SR-
BII overexpressed in nonphagocytotic human epithelial cells
have been shown to mediate the binding and uptake of live, as
well as dead, gram-negative and gram-positive bacteria, sug-
gesting a conserved role for SR-BI and SR-BII in pattern
recognition and host defense (50, 60). Intracellular signaling
pathways activated by pattern recognition receptors have been
shown to dictate the maturation profile of DCs. To study
whether SR-BI is involved in DC maturation, we purified IgG
from anti-SR-BI serum and assessed the influence of the anti-
SR-BI antibody on the DC maturation state. As shown in Fig.
3A, purified anti-SR-BI IgG interacted strongly with SR-BI on
the surface of immature DCs. Then, immature DCs were ex-
posed to anti-SR-BI IgG for 16 h and the activation of imma-
ture DCs was measured by flow cytometry of defined cell
surface markers. Whereas LPS induced DC maturation, the
exposure of DCs to anti-SR-BI IgG did not result in the up-
regulation of costimulatory molecules (Fig. 3B). Interestingly,
similar results were reported for DCs exposed to anti-DC-
SIGN antibodies (12, 28). These data indicate that the binding
FIG. 3. Binding of anti-SR-BI IgG and DC activation. (A) Cell surface expression of SR-BI detected by purified anti-SR-BI IgG. Cells were
incubated with purified anti-SR-BI IgG or purified preimmune control IgG (CTRL IgG) and subsequently stained with PE-conjugated anti-rat
IgG. Cells stained with the secondary antibody alone served as negative controls (gray-shaded curve [NC]). (B) Anti-SR-BI IgG and DC activation
by anti-SR-BI IgG. Immature DCs were exposed to purified anti-SR-BI IgG, purified CTRL IgG (50 ?g/ml each), or LPS (10 ?g/ml). After 16 h,
DC activation by purified anti-SR-BI IgG, CTRL IgG, or LPS was assessed by flow cytometric analysis of HLA-DR, CD80, CD86, and CD83 cell
surface expression (dark lines). Histograms corresponding to background expression of the respective cell surface molecules in unexposed DCs are
shown as gray lines. A result representative of three independent experiments using immature DCs from three different donors is shown. FL2-H,
VOL. 82, 2008SR-BI AND HCV CROSS-PRESENTATION 3469
of anti-SR-BI IgG to cell surface SR-BI may be not sufficient
to induce DC maturation. However, we cannot exclude the
possibility that SR-BI is capable of modulating intercellular
signals originated from other maturation-inducing factors, as
shown for DC-SIGN (12).
At an immature stage, DCs are characterized by their high
ability to capture antigens. We have previously shown that
immature DCs bind and rapidly internalize HCV-LPs in a
concentration-dependent manner. To study whether cell-sur-
face SR-BI expression correlates with the ability of DCs to
capture viral antigens, we determined SR-BI expression and
HCV-LP binding to DCs during the differentiation of mono-
cytes into DCs. Indeed, SR-BI expression continuously in-
creased during the differentiation of monocytes into DCs (Fig.
4A) and correlated with the initiation of HCV-LP binding (Fig.
4B). This correlation suggested that SR-BI may be involved in
HCV-LP binding to DCs. To confirm the role of SR-BI in the
binding of HCV antigen, we analyzed the binding of HCV-LPs
to DCs in the presence of anti-SR-BI targeting the extracellu-
lar loop of SR-BI. Preincubation of DCs with anti-SR-BI in-
hibited HCV-LP binding to DCs in a concentration-dependent
manner (Fig. 5A and B). By contrast, preincubation of DCs
with anti-CD36 did not affect HCV-LP binding (Fig. 5C). To
confirm that the inhibition of HCV-LP binding was indeed
mediated by anti-SR-BI antibodies, we assessed HCV-LP
binding to DCs in the presence of purified IgG from both
anti-SR-BI serum and control serum. Purified anti-SR-BI IgG
inhibited HCV-LP binding in a manner similar to anti-SR-BI
serum: the inhibition of HCV-LP binding in the presence of
anti-SR-BI IgG (100 ?g/ml) was 64% compared to the inhibi-
tion of its binding to DCs in the presence of control IgG (100
?g/ml). These data indicate that SR-BI plays a crucial role in
mediating the binding of HCV particles to DCs. A key role of
SR-BI for HCV binding to DCs is supported by two further
observations: (i) HCV-LP binding to monocytes is weak de-
spite a high level of expression of CD36 (Fig. 2 and 4B) and (ii)
only cells transfected with the human SR-BI, but not CD36,
resulted in recombinant HCV envelope glycoprotein E2 bind-
The ability of transfected or retrovirally delivered synthetic
small interfering RNAs to block the expression of specific
transcripts has proved useful for the analysis of gene function
in mammalian cells. However, since the efficient and sustained
delivery of small interfering RNA into DCs was not easily
achievable (data not shown), we assessed the role of SR-BI for
HCV antigen recognition by the ability of SR-BI ligands to
block HCV-LP binding. Like most SRs, SR-BI recognizes a
wide range of ligands, including polyanionic molecules, native
HDL, LDL, and very-low-density lipoprotein, as well as vari-
ous chemically modified HDL and LDL species. Distinct li-
gand-binding sites for HDL and LDL have been reported to
exist on SR-BI (26), indicating distinct modes of binding and
perhaps distinct binding sites for the various SR-BI ligands.
Recently, HDL and human serum have been shown to enhance
HCV pseudoparticle (HCVpp) and HCVcc infectivity (6, 19,
34, 43, 61), whereas oxidized LDL inhibited HCVpp and
HCVcc infectivity of human hepatoma cells (62). To study
whether similar mechanisms operate in DCs, we analyzed
HCV-LP binding to DCs in the presence of lipoproteins. As
shown in Fig. 5D, oxidized LDL reduced HCV-LP binding to
DCs to about 40%, while native LDL had no effect. By con-
trast, HDL enhanced HCV-LP binding to DCs by about four-
fold (Fig. 6A), similar to human serum (Fig. 6B). The mech-
anism by which lipoproteins modulate HCV infectivity is still
unclear. It is possible that distinct lipoproteins induce confor-
mational changes of the HCV particles and/or that the lipopro-
tein–SR-BI interaction may modulate virus binding and entry.
Interestingly, the presence of fucoidan, a nonspecific inhibitor
that blocks lipoprotein uptake by class A and B SRs (29) and
has been previously shown to interfere with the binding and
uptake of mycobacteria in monocyte-derived macrophages
(71), reduced HCV-LP binding to DCs by up to 90% (Fig. 5D).
Furthermore, in the presence of anti-SR-BI, the HDL-medi-
ated enhancement of HCV-LP binding to DCs was reversed
(Fig. 6C). Taken together, these data suggest that SR-BI is
involved in HCV binding to DCs.
SR-BI is a host entry factor for HCV-LP uptake into DCs.
Since SR-BI has been shown to represent a cellular cofactor
for HCVcc infection in human hepatoma cells (25, 31, 69), we
analyzed the role of SR-BI in HCV-LP uptake by DCs by
immunofluorescence and confocal LSM. First, to demonstrate
FIG. 4. SR-BI expression correlates with HCV-LP binding during
DC differentiation. Analysis of SR-BI cell surface expression (A) and
HCV-LP binding during differentiation of monocytes into DCs (B).
Monocyte-derived DCs were harvested at different time points during
culture in cytokine-conditioned medium. Then, monocytes and DCs
were analyzed for SR-BI expression and HCV-LP binding. Expression
of SR-BI was determined by flow cytometry using anti-SR-BI poly-
clonal serum as described in the Fig. 1 legend for panel C. HCV-LP
binding to DCs was determined by flow cytometry using a monoclonal
anti-HCV E2 antibody and PE-conjugated anti-mouse IgG. Data are
shown as net mean fluorescence intensity (? MFI) of a representative
3470 BARTH ET AL. J. VIROL.
that intact HCV-LPs are taken up by DCs, we used LSM to
visualize the HCV core protein, as well as the two envelope
glycoproteins E1 and E2, inside these cells (Fig. 7A). By dou-
ble staining core and E1 or core and E2, we showed that these
proteins colocalized both on the cell surface of DCs in binding
experiments and inside DCs when HCV-LPs were allowed to
enter DCs at 37°C (Fig. 7B). These findings demonstrate that,
indeed, particular structures containing the HCV structural
proteins are internalized. To study whether HCV-LP uptake is
mediated by envelope glycoprotein E2, HCV-LPs were prein-
cubated with an antibody directed against envelope glycopro-
tein E2 prior to incubation with DCs. As shown in Fig. 7C and
D, preincubation of HCV-LPs with anti-E2 antibodies signifi-
cantly inhibited HCV-LP uptake. These data demonstrate that
the uptake of HCV-LPs by DCs is mediated at least in part by
E2-cell surface protein interactions.
Next, we studied SR-BI expression by immunofluorescence
using anti-SR-BI antibody, as described in the Fig. 1 legend. As
shown in Fig. 8A, LSM of DCs incubated with anti-SR-BI
demonstrated the expression of SR-BI on the DC surface.
Next, to study HCV-LP uptake, DCs were preincubated with
anti-SR-BI prior to the addition of HCV-LPs. As shown in Fig.
8B, the incubation of DCs with HCV-LPs at 4°C in the pres-
ence of preimmune serum resulted in the detection of HCV-
LPs exclusively on the cell surface, consistent with HCV-LP
binding to the DC surface. The incubation of DCs with HCV-
FIG. 5. HCV-LP binding to human DCs is mediated by SR-BI. (A) DCs were preincubated with anti-SR-BI, preimmune serum, or PBS, and
HCV-LP binding to DCs was determined by flow cytometry using a monoclonal anti-HCV E2 antibody and PE-conjugated anti-human IgG. The
negative control (NC) histograms represent the results for DCs incubated with an insect cell control preparation. The x and y axes show mean
fluorescence intensities and relative numbers of stained cells, respectively. (B) Concentration-dependent inhibition of HCV-LP binding to DCs by
anti-SR-BI. Values are shown as net mean fluorescence intensity (? MFI) of duplicate measurements. (C) Specific inhibition of cellular HCV-LP
binding by anti-SR-BI. Prior to the addition of HCV-LPs, DCs were preincubated with anti-CD36, anti-SR-BI, anti-CD81, control IgG, or
preimmune serum. Cellular HCV-LP binding was determined as described above. Data are shown as percent HCV-LP binding (means ? standard
deviations of the results from three experiments) relative to HCV-LP binding in the absence of antibodies (100%). (D) Inhibition of cellular
HCV-LP binding by SR-B ligands. HCV-LP binding to DCs was determined in the presence of SR ligands fucoidan (1 ?g/ml) and oxidized LDL
(10 ?g/ml) or the control ligands poly(C) (1 ?g/ml) and LDL (10 ?g/ml). Data are shown as percent HCV-LP binding (means ? standard
deviations of the results from three independent experiments) relative to HCV-LP binding in the absence of ligands (100%). FL2-H, fluorescence
VOL. 82, 2008 SR-BI AND HCV CROSS-PRESENTATION3471
LPs at 37°C following preincubation of DCs with preimmune
serum resulted in the translocation of E2 immunoreactivity
into the cell, which is consistent with HCV-LP entry (Fig. 8C).
By contrast, the binding and uptake of HCV-LPs into DCs
were markedly inhibited by anti-SR-BI (Fig. 8B and C), indi-
cating that SR-BI is required for HCV-LP binding and uptake
into DCs. Of note, the anti-SR-BI serum used, as well as
purified anti-SR-BI IgG, have been shown to specifically in-
hibit HCVcc infection of human hepatoma cells (69), suggest-
ing that the uptake of HCV-LPs into DCs, as well as HCVcc
infection of hepatoma cells, may be mediated by similar SR-
BI–HCV envelope interactions.
SR-BI-mediated HCV-LP uptake results in trafficking of
viral antigens to the MHC class I pathway. Bacterial uptake by
nonphagocytotic human epithelial cells overexpressing SR-BI
has been shown to colocalize with cytosolic polyubiquitins and
proteasome (60). Moreover, macrophages from SR-BI-knock-
out mice showed a reduced cytosolic bacterial accumulation
(60), suggesting that SR-BI mediates bacterial recognition and
processing through a proteasome-dependent mechanism.
Since the uptake of HCV-LPs leads to an efficient processing
and presentation of HCV-LP-derived peptides on MHC class
I molecules (5), we studied HCV-LP cross-presentation in the
presence of anti-SR-BI and SR-BI ligands. DCs were preincu-
bated with anti-SR-BI or preimmune serum prior to the addi-
tion of HCV-LPs. Then, HCV-LP-pulsed DCs were matured
overnight with CD40L. After being washed, the DCs were
cocultured with HCV core-specific CD8?T cells. As shown in
Fig. 9A and B, anti-SR-BI serum markedly inhibited the IFN-?
production of HCV core-specific CD8?T cells in comparison
to the results for preimmune serum. A similar inhibition of the
IFN-? production of HCV core-specific CD8?T cells was
observed when DCs were incubated with the SR-BI ligand
fucoidan (Fig. 9B). By contrast, preincubation of DCs with
control ligand poly(C) did not affect HCV-LP cross-presenta-
tion (Fig. 9B). These findings indicate that SR-BI may target
viral antigens into the cytosol, where the viral antigens gain
access to the MHC class I presentation pathway.
Recently, HCVcc infection of human hepatoma cells has
been shown to depend on cholesterol and the cooperation
between SR-BI and CD81 (31). CD81 belongs to the tet-
raspanin family. These proteins associate with partner proteins
and facilitate their lateral positioning in the membrane, which
in turn affects the association with molecules involved in intra-
cellular signaling (37). Even though CD81 is highly expressed
on the DC surface (Fig. 2) (41), HCV-LP binding (Fig. 5C) and
cross-presentation (Fig. 9B) were not inhibited by anti-CD81.
Furthermore, T cells with a high level of expression of CD81
and no expression of SR-BI (Fig. 2) did not bind HCV-LPs (5).
These data suggest that, in contrast to SR-BI, CD81 appears
not to play a key role for HCV uptake and presentation in DCs
in our model system.
SR-BI is a host entry factor for tissue culture-derived HCV
uptake into DCs. Aiming to study whether SR-BI mediates the
uptake of HCVcc, we analyzed the uptake of iodixanol gradi-
ent-purified HCVcc into DCs in the presence of purified anti-
SR-BI IgG or control IgG. HCVcc uptake into DCs was ana-
lyzed by anti-E2-specific immunofluorescence and LSM. First,
to demonstrate that HCVcc are taken up by DCs, we used
LSM to visualize the HCVcc envelope protein E2 inside the
FIG. 6. HCV-LP binding to human DCs is enhanced by HDL.
(A) Enhancement of HCV-LP binding to DCs by HDL. HCV-LPs
were preincubated for 1 h at room temperature with different concen-
trations of HDL (diamonds) and LDL (triangles). After the addition of
HCV-LP–lipoprotein complexes to the DCs, HCV-LP binding was
determined as described in the Fig. 4 legend for panel A. Data are
shown as percent HCV-LP binding (means ? standard deviations of
the results from three experiments) in the presence of lipoproteins
compared to HCV-LP binding in the presence of PBS (100%). (B) En-
hancement of HCV-LP binding in the presence of lipoproteins present
in human serum. HCV-LPs were preincubated with human serum
from a healthy individual at the concentrations indicated and then
added to DCs at 4°C, allowing HCV-LP binding. (C) HDL-mediated
enhancement of HCV-LP binding is reversed by anti-SR-BI antibod-
ies. HCV-LPs were incubated with HDL (10 ?g cholesterol/ml or 50
?g cholesterol/ml) for 1 h at 37°C, while DCs were preincubated with
or without anti-SR-BI serum (1:20) for 1 h at room temperature.
Following the addition of HCV-LP–lipoprotein complexes to DCs
incubated with anti-SR-BI or control, HCV-LP binding was deter-
mined using mouse anti-E2 MAb (AP33) as described above. Data are
shown as percent HCV-LP binding (means ? standard deviations of
the results from three independent experiments) relative to HCV-LP
binding in the absence of ligands (100%).
3472 BARTH ET AL.J. VIROL.
FIG. 7. HCV-LP uptake into DCs is mediated by envelope glycoprotein E2. (A) HCV-LP uptake by DCs. DCs were incubated with HCV-LPs
or insect cell control preparations (GUS) and triple stained for actin (green); viral protein core, E1, or E2 (red); and nucleus (DAPI [4?,6?-
diamidino-2-phenylindole], in blue). Arrows indicate viral protein staining. (B) HCV-LPs internalized in DCs. DCs incubated with HCV-LPs were
triple stained for nucleus (DAPI, in blue), core (green), and E1 or E2 (red). Overlay of images shows colocalization of core/E1 or core/E2 (right
panel). (C) HCV-LP uptake by DCs is mediated by envelope glycoprotein E2. HCV-LPs were preincubated (1 h at 37°C) with anti-E2 antibody
(AP33; 50 ?g/ml) or control IgG (50 ?g/ml) before incubation with DCs. HCV-LP–anti-E2 complexes were then added to DCs and incubated at
37°C for 3 h. Following fixation, DCs were triple stained for actin (green), E2 (red), and nucleus (DAPI, in blue). (D) Quantitation of HCV-LP
uptake in the presence and absence of anti-E2 antibody. HCV-LP uptake by DCs in the presence of anti-E2 MAb or control IgG is shown as
percentage of cells with positive intracellular HCV-LP E2 staining relative to the total number of cells. The means ? standard deviations of the
results from three independent experiments are shown. Statistical analysis was performed by Student’s t test.
cells. As shown in Fig. 10A, HCVcc envelope glycoprotein E2
colocalizes with the cytoplasm of DCs following an incubation
step of DCs with HCVcc at 37°C. Interestingly, only about 8 to
15% of DCs incubated with HCVcc stained positive for HCV
E2 protein. In contrast, no internalization of HCVcc E2 pro-
tein was observed when DCs were incubated with HCVcc at
4°C (data not shown). These findings demonstrate that
HCVcc-derived envelope glycoprotein E2 is internalized into
DCs in a temperature-dependent manner. To study whether
HCVcc uptake is mediated by SR-BI, DCs were preincubated
with purified anti-SR-BI IgG or control IgG. As shown in Fig.
10, purified anti-SR-BI IgG markedly and significantly inhib-
ited HCVcc uptake into DCs, whereas purified control IgG
had no effect. These data demonstrate that the uptake of
HCVcc by DCs is mediated at least in part by SR-BI and that
SR-BI most likely represents a host entry factor for the uptake
of infectious HCV into DCs.
In this study, we assessed the functional role of SR-BI for
the uptake and cross-presentation of HCV by human DCs. We
demonstrate that (i) SR-BI is required for the binding and
uptake of HCV by human DCs and (ii) SR-BI-mediated up-
take results in trafficking into the MHC class I pathway, fol-
lowed by efficient cross-presentation to HCV-specific CD8?
T-cells. Taken together, our results reveal a novel function for
SR-BI for antigen uptake and presentation and identify a novel
mechanism whereby DCs can capture and process viral anti-
SR-BI and its splicing variant SR-BII are physiologically
relevant HDL receptors with an identical extracellular loop.
SR-BII differs from SR-BI at the C terminus, which is reported
to confer an intracellular localization on SR-BII (64). Using
defined antibodies targeting the cytoplasmic tail or extracellu-
lar loop of SR-BI, we could show that human immature DCs
express SR-BI. These findings are in line with the results of two
previous studies demonstrating that SR-BI is expressed on
monocyte-derived DCs, as well as on plasmacytoid and my-
eloid DCs (10, 67). In contrast to our findings, Yamada et al.
(67) observed a higher level of SR-BI expression on the surface
of monocytes using a different anti-SR-BI antibody. These
differences could be due to different epitopes recognized by the
antibodies or different protocols of monocyte isolation used in
their study and ours. In our study, as well as in the study of
Buechler et al. (10), SR-BI expression was induced during the
differentiation of monocytes into DCs, indicating that SR-BI
may play a specific role for DC function. Since SR-BI has been
shown to represent a host cell entry factor for HCV infection
of human hepatoma cells (25, 31, 69), we explored its role in
viral antigen capture and presentation by DCs. Using an HCV-
LP-based model system (5), we demonstrate that SR-BI is
required for the binding and uptake of HCV-LP into DCs.
Since previous results have shown that C-type lectins, such as
mannose receptor or DC-SIGN, were not sufficient to mediate
HCV-LP binding to DCs (5), SR-BI may represent one of the
key DC surface proteins binding HCV particles on DCs. This
novel SR-BI function is further supported by the observation
that HDL enhanced the binding of HCV-LP to DCs, whereas
oxidized LDL and polyanionic ligands reduced HCV-LP bind-
ing. Since the presence of HDL did not inhibit but rather
enhanced HCV-LP binding, it is unlikely that HCV and HDL
compete for the SR-BI HDL binding domain. The highly re-
producible enhancement of HCV-LP binding by HDL may
rather point to a more-efficient interaction of SR-BI with
HCV, e.g., as a result of a conformational change induced by
HDL. These findings are in line with findings observed for the
infection of human hepatoma cells with recombinant HCVpp
FIG. 8. SR-BI mediates HCV-LP uptake into DCs. (A) SR-BI ex-
pression on the DC surface. DCs were incubated with anti-SR-BI or
preimmune serum (1:10 dilution). After being washed with PBS, DCs
were incubated with fluorescein isothiocyanate-conjugated anti-mouse
IgG. Microphotographs illustrate SR-BI expression after incubation
with preimmune serum (left panel) or anti-SR-BI (right panel). Nu-
clear staining (DAPI [4?,6?-diamidino-2-phenylindole]) is shown in
blue. (B) For determination of HCV-LP binding, DCs were incubated
with HCV-LPs at 4°C after preincubation of DCs with preimmune
serum (left panel) or anti-SR-BI (right panel). Cell-bound HCV-LPs
were detected by immunofluorescence using a monoclonal anti-HCV
E2 antibody (red fluorescence). For costaining of cytoplasmic struc-
tures, cells were coincubated with an antiactin antibody (green fluo-
rescence). (C) For determination of HCV-LP uptake, DCs were incu-
bated with HCV-LPs at 37°C after preincubation of DCs with
preimmune serum (left panel) or anti-SR-BI (right panel) and ana-
lyzed as described above. Arrows indicate HCV-LP staining.
3474 BARTH ET AL. J. VIROL.
and HCVcc (6, 19, 34, 43, 61). The significant modulation of
HCV-LP binding by HDL and LDL provides a link between
lipid metabolism and antigen recognition and may suggest that
lipoproteins may interfere with the DC-antigen interaction.
Antigen cross-presentation offers a solution by permitting
DCs to crossover exogenous antigens for access to the class I
MHC peptide-loading machinery. This mechanism enables
DCs to raise immune responses against pathogens, like viruses,
that do not infect them (1). Since robust HCV infection of DCs
has not been documented either in vivo (49) or in vitro (17), it
is likely that the cross-presentation of HCV antigens repre-
sents an important mechanism for the induction of antiviral
CD8?T-cell responses. This hypothesis is further supported by
our data clearly demonstrating that productive infection of
FIG. 9. SR-BI is involved in HCV-LP cross-presentation to HCV-specific CD8?T cells. (A) HCV-LP cross-presentation in the presence of
anti-SR-BI antibody. DCs were incubated with anti-SR-BI, control serum, or lactacystin prior to the addition of HCV-LPs, as described in Material
and Methods. DCs incubated with HCV core peptide core36-53 or an insect cell lysate control preparation (GUS) served as positive and negative
controls, respectively. After 24 h, DCs were cocultured with autologous HCV core-specific CD8?T cells (recognizing an epitope in the HCV core
protein comprising amino acids 36 to 53) and analyzed by flow cytometry after staining with antibodies to CD8 and IFN-?. The percentages of
CD8?T cells that produced IFN-? in the respective quadrants are indicated on the dot plots. FITC, fluorescein isothiocyanate. (B) HCV-LP
cross-presentation in the presence of SR-B ligands, anti-SR-BI, and anti-CD81. Data are shown as percent HCV-LP cross-presentation relative
to HCV-LP cross-presentation in the absence of the respective antibodies or SR-BI ligands (100%). Mean percentages ? standard deviations of
the results of three independent experiments are shown for anti-SR-BI and preimmune serum. Statistical significance of differences between DCs
preincubated with anti-SR-BI and control serum was determined by the two-tailed t test.
VOL. 82, 2008 SR-BI AND HCV CROSS-PRESENTATION3475
DCs is not required for efficient HCV antigen presentation.
This observation extends previous findings for human immu-
nodeficiency virus (HIV). DCs efficiently cross-present HIV
antigens captured from both live and apoptotic infected CD4?
T cells, whereas HIV presentation after direct infection of DC
was not detectable even with a high amount of replicative virus
(42). Since HCV does not replicate efficiently in DCs (49), it is
likely that the acquisition of HCV antigens for cross-presenta-
tion by SR-BI might be a critical point for the development of
an early immune response at the early stages of HCV infection.
However, the development of a strong T-cell immunity is re-
stricted to antigen-capturing DCs which have been exposed to
a stimulus that leads to their maturation. We have previously
demonstrated that HCV-LPs induce a small but significant
upregulation of the costimulatory molecules CD80 and CD83
(5). In this study, HCV-LP-pulsed DCs were stimulated with
CD40L overnight to ensure sufficient DC maturation. In vivo
studies suggest that CD40 is provided by NK lymphoctes in an
early DC-NK lymphocyte interaction (21). Since this interac-
tion likely takes place at the site of infection and in secondary
lymphoid organs, the maturation of HCV-LP-pulsed DCs by
CD40L could reflect the scenario for antigen presentation in
an acute HCV infection.
HCV-LP cross-presentation was markedly inhibited in the
presence of anti-SR-BI, suggesting that SR-BI is involved in
the trafficking of viral antigens toward the MHC class I path-
way. This finding suggests that SR-BI may act as an immuno-
receptor facilitating the intracellular accumulation of viral an-
tigens and triggering processing and cross-presentation. This
hypothesis is further supported by recent data demonstrating
that SR-BI mediates bacterial adhesion and cytosolic accumu-
lation (60). Moreover, other members of the growing SR fam-
ily, SR-A and LOX-1, have been shown to be involved in the
uptake and trafficking of exogenous antigens toward the MHC
class I pathway (18, 27). Since the anti-SR-BI antibody used in
this study may also target the large extracellular loop of SR-
BII, we cannot exclude a role for SR-BII in viral antigen
uptake and cross-presentation.
Interestingly, HCV-LP cross-presentation could not be com-
pletely inhibited by anti-SR-BI, suggesting that additional re-
ceptors are involved in targeting HCV-LPs into the MHC class
I pathway. Recent studies have shown that the initiation of
HCV infection is dependent on a cooperativity between SR-BI
and CD81 (31). In contrast to the findings for HCVcc infec-
tion, CD81 did not appear to play a major role in HCV-LP
binding and cross-presentation in DCs. These data suggest that
SR-BI is the main HCV capture receptor on DCs, while a
cooperative action of SR-BI and CD81 is required for efficient
HCV infection of hepatocytes. Furthermore, these data illus-
trate the difference in HCV entry pathways in hepatocytes and
DCs. In hepatocytes, HCV enters by clathrin-mediated endo-
cytosis, followed by an HCV envelope membrane fusion pro-
cess for the delivery of the HCV genome into the cytosol (3, 8).
In contrast, classical MHC class I presentation requires the
transfer of the exogenous antigens from the endosome or
phagosome into the cytosol, where the antigens are degraded
by proteasomes into oligopeptides. The peptides are then
transported by the transporter associated with antigen process-
ing into the endoplasmic reticulum and are bound to MHC
class I molecules. In an alternative pathway, peptides may be
generated within the endocytotic compartment and the result-
ing peptides are then bound to recycling MHC class I mole-
cules (1). Further studies analyzing the molecular mechanisms
of HCV-LP processing and presentation are in progress. Pre-
liminary studies demonstrated that lactacystin, a highly specific
inhibitor of proteasomal antigen processing, did not inhibit
HCV-LP cross-presentation (Fig. 9A). These results may indi-
cate that alternative MHC class I processing and presentation
pathways could be involved in HCV-LP cross-presentation or
that additional, as-yet-unidentified cytosolic proteases down-
stream of the proteasome could participate in HCV-LP pro-
cessing and presentation. Interestingly, several viral epitopes
have been identified that are produced or presented more
efficiently when proteasome activity is impaired or altered,
including viral epitopes from influenza virus (39, 66) and HIV
(14). Studies are under way to analyze these mechanisms in
FIG. 10. SR-BI mediates HCVcc uptake into DCs. (A) For analysis
of HCVcc entry, DCs were incubated with PBS (left panel) or iodixa-
nol gradient-purified JFH1 HCVcc (right panels) at 37°C as described
in Materials and Methods. Internalized HCVcc were detected by im-
munofluorescence using a monoclonal anti-HCV E2 antibody (red
fluorescence). For costaining of cytoplasmic structures, cells were co-
incubated with an antiactin antibody (green fluorescence). The nucleus
is stained with DAPI (4?,6?-diamidino-2-phenylindole) (blue fluores-
cence). Arrows indicate HCVcc E2 protein. To study whether HCVcc
uptake is mediated by SR-BI, DCs were preincubated with purified
anti-SR-BI IgG or control IgG as described in Materials and Methods.
(B) HCVcc uptake was quantified by counting the average number of
cells with positive staining for HCVcc E2 protein per total cells (n ?
300) in the presence or absence of purified anti-SR-BI IgG or control
IgG. Results shown are the means and standard deviations of the
results of three independent experiments (from three different DC
preparations and two donors) performed in duplicate (number of
HCV E2-positive cells for DCs incubated with HCVcc in the absence
of purified antibody, 100%). Statistical significance of differences be-
tween the number of E2-positive DCs following preincubation with
purified anti-SR-BI IgG compared to DCs preincubated with purified
control IgG was determined by the two-tailed t test.
3476BARTH ET AL. J. VIROL.
In this study, we used an HCV-LP-based model system to
assess the molecular mechanisms of HCV particle uptake and
presentation by human DCs (5). HCV-LPs are generated by
self-assembly of HCV structural proteins in insect cells (7) and
are characterized by morphological, biophysical, and antigenic
properties similar to those of infectious virions (22, 63). Fur-
thermore, the binding and uptake of HCV-LPs to target cells
appear to require a set of viral epitopes and cellular host
factors similar to that required by infectious HCV (2, 4, 55, 59).
Although we cannot exclude the possibility that the virus-like
particle concentration in our in vitro experiments may exceed
the concentration of circulating infectious viral particles inter-
acting with DCs in vivo, studies in animal models, including
mice and chimpanzees, have shown that HCV-LPs used in
amounts as in this study are appropriate for HCV-LP uptake
and presentation by DCs in vivo. Indeed, in vivo studies have
demonstrated that HCV-LPs induce a strong antiviral humoral
and cellularimmune response,
T-helper cells and cytotoxic T lymphocytes, in primates, includ-
ing chimpanzees (30, 35, 46, 51). The quantity and quality of
HCV-LP-induced cellular immune responses against the HCV
structural proteins appear to be similar to the immune re-
sponses induced by the infectious virus (30, 35, 46, 51). More-
over, HCV-LP-induced T-cell responses result in control of
HCV infection in the chimpanzee in vivo (20). These findings
and the successful use of virus-like particles of other viruses,
including HIV (11), hepatitis B virus (56), papillomavirus (36,
53), and parvovirus (44), for the study of virus uptake and
antigen presentation in DCs indicate that the interaction of
HCV-LPs with DCs represents an appropriate model system to
study the molecular mechanisms of HCV particle uptake and
presentation of HCV structural proteins.
To confirm the validity of the HCV-LP model system, as well
as the role of SR-BI for HCV uptake into DCs, we produced
high-titer, gradient-purified HCVcc and studied HCVcc up-
take by using anti-E2-specific immunofluorescence and confo-
cal LSM. Using this method and purified anti-SR-BI IgG,
recently shown to inhibit HCVcc infection of hepatoma cells
(69), we demonstrate that anti-SR-BI IgG specifically inhibits
the uptake of HCVcc into DCs (Fig. 10). These findings dem-
onstrate the relevance of the HCV-LP model system for the
study of HCV particle uptake and confirm the specificity of the
anti SR-BI serum used for the study of HCV-DC interaction.
In conclusion, we have demonstrated that SR-BI mediates
HCV-LP and HCVcc uptake into human DCs, indicating that
SR-BI may represent a cell-surface receptor for the recogni-
tion of viral antigens. The inhibition of HCV-LP cross-pre-
sentation by anti-SR-BI antibody suggests that SR-BI is
implicated in trafficking exogenous viral antigens toward the
MHC class I presentation pathway. Taken together, these
findings support a novel function of SR-Bs for viral antigen
uptake and recognition. In addition, the SR-BI–viral antigen
interaction may represent a novel target for therapeutic or
preventive strategies aiming at the induction of efficient
antiviral immune responses.
The authors thank Natalie Wischniowski (Department of Medicine
II, University of Freiburg, Germany) and Isolde Friedrich (Divison of
Clinical Chemistry, Department of Medicine, University of Freiburg,
Germany) for excellent technical assistance in the analysis of HCV-LP
cross-presentation and purification of lipoproteins, respectively. We
thank Erik Depla (Innogenetics, N.V., Ghent, Belgium), H. B. Green-
berg (Division of Gastroenterology, Department of Medicine, Stan-
ford University School of Medicine, Palo Alto, CA) and Arvind Patel
(MRC Virology Unit, Glasgow, United Kingdom) for the kind gift of
MAbs and F. Stoll-Keller and G. Inchauspe ´ for helpful discussions.
This work was supported by the German Research Foundation,
Bonn, Germany (DFG Ba1417/11-1 and 11-2 to T.F.B. and TH791/2-3
to R.T.), the European Union (EU NoE VIRGIL, LSHM-CT-2004-
503359 to T.F.B.), the German Ministry for Education and Research,
Berlin, Germany (BMBF 01K19951 to T.F.B.), the German Liver
Foundation, Hannover, Germany (T.F.B), Inserm, ANR (ANR-05-
CEXC-008), and ANRS (grant no. 06221), Paris, France (T.F.B.), and
the Intramural Research Program of the National Institute of Diabetes
and Digestive and Kidney Diseases, NIH (H.B., Z.H., and T.J.L.).
M.B.Z. was supported by the Inserm Poste Vert program in the frame-
work of the Inserm European Associated Laboratory Freiburg-Stras-
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
The authors declare that no competing interests exist.
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