1 The Edward Jenner Institute for Vaccine Research, Compton, Newbury RG20 7NN, UK
2 Institute of Medical Virology, Justus Liebig University, Giessen, Germany
3 Division of Microbiology and Infectious Diseases, The University of Nottingham, Nottingham, UK
4 Istituto di Ricerche di Biologia Moleculare P. Angeletti, Rome, Italy
5 CNRS-UPR2511, Institut Pasteur de Lille, Lille, France
6 MRC Virology Unit, Institute of Virology, Glasgow, UK
Correspondence
Persephone Borrow
persephone.borrow{at}jenner.ac.uk
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ABSTRACT |
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Supplementary material is available in JGV Online.
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MAIN TEXT |
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HCV attachment/entry into host cells is thought to be mediated by the envelope glycoproteins, E1 and E2, which form non-covalent heterodimers. Cell-surface molecules reported to bind E2 include CD81 (Pileri et al., 1998), scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002
), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and liver/lymph node (L)-SIGN (Gardner et al., 2003
; Lozach et al., 2003
; Pohlmann et al., 2003
), and glycosaminoglycans (GAGs) (Takikawa et al., 2000
; Yagnik et al., 2000
; Basu et al., 2004
). Of these, DC-SIGN, L-SIGN and GAGs have been suggested to function as capture receptors that facilitate HCV interaction with specific entry receptors (Gardner et al., 2003
; Pohlmann et al., 2003
; Basu et al., 2004
; Lozach et al., 2004
), whereas CD81 and SR-BI are thought to act as (co-)receptors that mediate HCV binding and subsequent cell entry and infection (Bartosch et al., 2003b
; Zhang et al., 2004
). HCV particles associated with low-density lipoprotein (LDL) may also enter cells using the LDL receptor (Agnello et al., 1999
; Wunschmann et al., 2000
).
E2receptor interactions may not only be involved in virion attachment/entry, but may also block or mimic receptor functions, contributing to viral persistence/pathogenesis. CD81, a member of the tetraspanin superfamily, has a variety of biological functions, which include the regulation of lymphocyte activation [CD81 cross-linking enhances T/B-cell responses to stimulation through their antigen-specific receptors and inhibits natural killer (NK) cell activation] (Levy et al., 1998; Crotta et al., 2002
; Tseng & Klimpel, 2002
). SR-BI, a member of the CD36 superfamily, acts as a high-density lipoprotein receptor, mediating cholesterol uptake (Krieger, 2001
), but also functions as a pattern-recognition receptor on monocytes and macrophages (Pearson, 1996
; Imachi et al., 2000
).
To gain insight into HCV interaction with haematopoietic cells and the potential for HCV to modulate the responses of PBMC subsets via receptor interaction, we analysed the expression of HCV (co-)receptors CD81 and SR-BI on PBMC subsets and characterized the binding of genotype 1 soluble truncated recombinant E2 (sE2) proteins to these cells.
PBMC subsets were identified using monoclonal antibodies (mAbs) against distinguishing surface markers (Supplementary Table S1). CD81 and SR-BI expression were analysed by co-staining with a FITC-conjugated anti-CD81 mAb (clone JS-81; PharMingen BD) or anti-SR-BI mAb [clone 3D5 (A. Vitelli and others, unpublished data)] followed by FITC-conjugated anti-mouse IgG/IgM F(ab')2 (Jackson Immuno Research). As expected, we observed CD81 expression on the hepatocyte cell line Hep3B and on PBMCs (Fig. 1a). Although all PBMC subsets expressed CD81, there was marked variation in the level of expression detected on different cell types (Fig. 1b
). NK, natural T (NT) and T cells expressed high levels of CD81. B cells, monocytes and myeloid DCs expressed intermediate levels of CD81, whilst only low levels of CD81 were detected on plasmacytoid DCs. High level SR-BI expression was observed on hepatocyte cell lines (including Hep3B; Fig. 1c
). It has previously been shown that SR-BI is expressed on monocytes and macrophages (Buechler et al., 1999
); here, we also documented its expression on plasmacytoid and myeloid DCs (Fig. 1c
). SR-BI was not detected on any other PBMC subsets.
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Notably, the PBMC subsets found to bind H77c E2660 via both CD81-dependent and CD81-independent mechanisms are those reported to be infected by HCV in vivo (Supplementary Table S1). Thus, although CD81 plays a prominent role in HCV binding to PBMC subsets, this interaction does not appear to be sufficient to allow infection of haematopoietic cells. This observation is in agreement with other studies suggesting that CD81 is required but not sufficient for the HCV attachment/entry process (Bartosch et al., 2003a, b
; Hsu et al., 2003
; Cormier et al., 2004
; McKeating et al., 2004
).
Most PBMC subsets exhibiting CD81-independent E2 binding expressed SR-BI; we thus investigated the role of SR-BI in CD81-independent E2 binding to monocytes (Fig. 2d). PBMCs were incubated with saturating quantities of anti-CD81 mAb (clone JS-81), anti-SR-BI mAb (clone 3D5), both mAbs or an isotype control mAb prior to the addition of sE2, and E2 binding to monocytes was analysed using Alexa Fluor 488-labelled mAb H53. The anti-CD81 mAb inhibited >50 % of E2 binding to monocytes. E2 binding was also partially inhibited by the anti-SR-BI mAb, showing that SR-BI played a role in E2 binding to monocytes. When used together, anti-CD81 and anti-SR-BI mAbs blocked H77c E2 binding to monocytes in an additive fashion, but a component of binding remained that could not be eliminated by saturating amounts of both mAbs. This residual binding may be mediated via DC-SIGN and/or GAGs, because a small proportion of CD14+ cells in peripheral blood express DC-SIGN (Turville et al., 2001
; Engering et al., 2002
) and GAGs are ubiquitously expressed (Kjellen & Lindahl, 1991
).
Whilst CD81 is proposed to have a critical role as an attachment receptor, SR-BI is a strong candidate to mediate HCV internalization into cells, as SR-BI internalizes its natural ligands to endosomal compartments (Silver & Tall, 2001; Bocharov et al., 2004
). Although SR-BI expression was detected on the majority of PBMC subsets reported to be infected by HCV in vivo, B cells constituted an exception to this; there may thus be an alternative pathway for virion internalization in these cells.
Although HCV infects haematopoietic cells, the liver constitutes the major site of in vivo virus replication. Further, the observation that retroviral particles pseudotyped with the HCV glycoproteins efficiently transduce hepatocytes but not PBMCs (Bartosch et al., 2003a; Cormier et al., 2004
) suggests that viral attachment/entry into PBMCs may be suboptimal. Our results show that at least some PBMC subsets are capable of binding E2 via CD81, SR-BI and additional receptor(s); but it is plausible that the level of expression of SR-BI on PBMCs may limit their susceptibility to infection, and/or that PBMCs lack expression of additional factor(s) required for efficient infection of hepatocytes.
Further experiments compared the binding of sE2s derived from H77c and other genotype 1 HCVs to PBMCs. sE2 proteins truncated at aa 661 (E2661) were produced from a second genotype 1a clone, UKN1A14, and two genotype 1b clones, BK and UKN1B12. BK is a well-characterized HCV clone (Scarselli et al., 2002). Plasmids encoding UKN1A14 and UKN1B12 E2661 were generated by subcloning aa 364661 (corresponding to the HCV-H strain) from E1E2 sequences amplified and cloned from patient serum (as described previously Lavillette et al., 2005
) into mammalian expression vector pcDNA3.1 (Invitrogen). Retroviral pseudoparticles expressing E1E2 proteins from these clones were shown to infect hepatocyte cell lines in vitro (Lavillette et al., 2005
), indicating the functionality of these E2 sequences. Supplementary Fig. S3 shows the sequences of the E2660/661 proteins used in this study. Quantities of each E2 protein normalized to contain equivalent amounts of monomeric E2 (Supplementary Fig. S4) were used in binding assays.
The binding of UKN1A14 and H77c E2660/661 to PBMCs was analysed using anti-E2 mAb H53 [which recognized both genotype 1a proteins equally well in a GNA lectin-capture enzyme immunoassay (EIA) (not shown)], followed by a FITC-conjugated secondary antibody (Fig. 3a). As H53 did not recognize genotype 1b BK and UKN1B12 E2s, an Alexa Fluor 488-conjugated anti-Penta-His mAb (Qiagen) was used for E2 detection in experiments addressing their binding to PBMCs (BK E2661 data are shown in Fig. 3b
; similar results were obtained with UKN1B12 E2661). UKN1A14, BK and UKN1B12 E2s were all found to exhibit a much lower level of binding to PBMCs than H77c E2660. This was likely a reflection of the relative abilities of these E2 proteins to bind to CD81: BK E2661 was previously reported to have a much lower CD81-binding affinity than H77 E2 (Scarselli et al., 2002
), and we also found that UKN1A14, BK and UKN1B12 E2661 proteins bound much less well than H77c E2660 to the CD81 large extracellular loop in EIAs (not shown).
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The variation in CD81-binding affinity of sE2 proteins from different viruses may not affect their infectivity, as retroviral particles pseudotyped with HCV glycoproteins from clones whose E2 proteins have both low and high CD81-binding abilities have been shown to be infectious in vitro (Bartosch et al., 2003b; McKeating et al., 2004
). However, the interaction of E2 with CD81 and other cell-surface proteins may not only be involved in viral entry but also in modulation of host-cell responses. Notably, it has been reported that E2 cross-linking of CD81 on lymphocyte subsets in vitro can modulate their response to activating stimuli in a manner analogous to CD81 cross-linking via anti-CD81 antibodies (Wack et al., 2001
; Crotta et al., 2002
; Tseng & Klimpel, 2002
), suggesting that E2CD81 interactions may modulate the host-immune response in vivo. If this is the case, it might be predicted that viruses bearing E2 proteins that bind with differential affinity to CD81 may have correspondingly different immunomodulatory capacities, potentially resulting in differences in persistence/pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 6 May 2005;
accepted 14 June 2005.