PLoS One. 2014 Jun 13;9(6):e99601.

The expression of the hepatocyte SLAMF3 (CD229) receptor enhances the hepatitis C virus infection.

Abstract

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

PMID: 24927415

 

Supplements:

Hepatitis C Virus (HCV) binding and entry into hepatocyte implicate SLAMF3 internalization machinery

Ingrid Marcq1, Hakim Ouled-Haddou1, Véronique Debuysscher1, Christèle Ossart2, Jean-Pierre Marolleau1, 2 and Hicham Bouhlal1, 2 *

1EA 4666-Centre Universitaire de Recherche en Santé CURS, CAP-Santé (FED 4231), Université de Picardie Jules Verne, CHU Sud, Amiens, France

2Service d’Hématologie Clinique – Laboratoire de Thérapie Cellulaire, Centre Hospitalier Universitaire Sud, Amiens, France

Conflict of interest statement: the authors have no financial conflicts of interest to declare.

Funding statement: project funding from the Conseil Régional de Picardie (reference: Imm-HCV) and CHU Amiens.

* Corresponding author: Hicham Bouhlal, EA 4666-LNPC, SFR CAP-Santé (FED 4231), Centre Universitaire de Recherche en Santé CURS-Université de Picardie Jules Verne and Centre Hospitalier Universitaire CHU Sud, Amiens, France; hicham.bouhlal@u-picardie.fr ; Phone: +33 3 22 82 79 13

 

Abstract

The Signaling Lymphocytic Activation Molecule SLAM-family proteins belong to immunoglobulins super family organized by two or four extracellular immunoglobulin (Ig)-like domains, a transmembrane domain and an intracytoplasmic region containing tyrosine-based motifs. SLAMF3, also named CD229, has four extracellular Ig-like domains; domains 1 and 3 are very similar, as are domains 2 and 4. To date, SLAMF3 expression has been documented in thymocytes, T cells, B cells, dendritic cells, macrophages and natural killer cells. During the formation of the immunological synapse between T cells and antigen-presenting B cells, SLAMF3 relocates to (and accumulates at) the edge of the contact area between the cells. Recently, we reported the expression of SLAMF3 by hepatocytes and this molecule is involved in the control of cell proliferation and in hepatocellular carcinogenesis. Furthermore, SLAMF3 expression was also found to be associated with inhibition of HCC xenograft progression in the nude mouse model (1). We also described the implication of SLAMF3 expressed by hepatocytes in the entry of Hepatitis C virus (HCV) and proposed that SLAMF3 could be a potential new target for developing antiviral drug. It has been shown by our group that SLAMF3 blockade by antibodies and down-modulation by siRNA decreases the susceptibility of hepatocytes to HCV and the ectopic expression of SLAMF3 increased infection (2). The SLAMF3 as newly identified HCV entry factor add complexity to the HCV entry process. Hepatitis C virus (HCV) entry into hepatocytes represents a complex mechanism dependent on several cellular and environmental factors such as pH and temperature. As summarized in figure 1, virus may also use glycosaminoglycans (GAGs) and low density receptors (LDL-R) as initial attachment factors. Tetraspanin CD81, scavenger receptor type I (SR-B1), and tight junction proteins Claudin-1 (CLDN1) and Occludin (OCLDN) are major late receptors known to mediate the entry of HCV. SLAMF3 is the only member of the SLAM family to interact with the µ-2 chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. More importantly, this interaction needs interaction between residue Tyrosine Y606 of SLAMF3 and Grb2 SH2 present mostly in lymphocytes and require receptor phosphorylation. The Grb2 binding site of SLAMF3 is important for the proper endocytosis of the receptor.

We hypothesize that the HCV entry throught SLMAF3 occur by endocytosis pathway.


Supplement

First, it is noteworthy that SLAMF3 is associated with the µ-2 chain of the AP-2 adaptor complex and is the only SLAM family member that can be internalized by clathrin-mediated endocytosis (3). It has been reported that SLAMF3 binds to the µ-2 chain of AP-2 complex. Analysis of SLMAF3 revealed only one sequence Y470EKL as the binding site to µ-2 chain of the AP-2. Using site-directed mutagenesis, Del Valle et al. mapped the µ-2 AP2 interaction to SLAMF3 in Y470 motif. The single replacement of tyrosine by phenylalanine abrogates µ-2 binding to SLAMF3 intracellular tail. In this model, the phenylalanine substitution abrogates the binding to µ-2 but leads to a weaker interaction and slower endocytosis. Thus, Tyr470 is essential for SLAMF3 endocytosis and may inhibit the internalization of this receptor after its phosphorylation in vivo (3). In T and B cells, SLAMF3 endocytosis is impaired when Fcγ Receptors are triggered and enhanced when the BCR is activated, this molecule may have a differential role in these different events. The ability to regulate SLAMF3 internalization differentially may allow for different net effects of SLAMF3 signaling which may be cell-specific. Other studies indicate that SLAMF3, unlike SLAMF1 and SLAMF5 (also named CD150 and CD84), inhibits interferon-g secretion after TCR activation in T cells (4).

On the other hand, SLAMF3, but not other members of the CD150 family, directly bind Grb2. Martin et al. reported that SLAMF3 mutant lacking the Grb2 binding site is not internalized after SLAMF3 engagement with specific antibodies. In addition, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired SLAMF3 endocytosis (5). Then in immune system, the rate of SLAMF3 endocytosis may be increased by TCR and BCR to prevent SLAMF3 from delivering negative signals to the cell and Fc-g receptor ligation retains the SLAMF3 on the cell surface, thus favoring signaling through this receptor and the maintenance of an inhibitory response. In hepatic system, the proven high level expression in healthy tissue compared to the low expression in cancerous cells Taken together, we hypothesized that HCV entry via SLAMF3 may use the endocytosis machinery of SLAMF3. We proved that the SLAMF3 overexpression in Huh-7 cell line significantly increased the entry of HCV into leading to infection of hepatocytes (2). To study the implication of SLAMF3-dependent endocytosis in HCV entry process, we used the HCV-refractory hepatocyte cell line, HepG2 that not allow achieving a complete viral cycle and therefore do not produce viral particles. In addition, HepG2 cells expressed low endogenous wild SLAMF3 (upto 3%) that reached 40% of positive cells after ectopic transfection (Figure 2). We generated two variants of SLAMF3 using site-directed mutagenesis where in the tyrosine Y470 and Y606 were changed to phenylalanine, the AP2 and Grb2 fixation sites, respectively at the intracytoplasmic tail of SLAMF3 (figure 3 a). Both SLAMF3 mutant forms, SLAMF3Y470F and SLAMF3Y606F, expressed at cell surface at similar level than SLAMF3WT, after ectopic introduction. Then, 38, 42 and 37% of cell show surface SLAMF3 expression in SLAMF3Y470F, SLAMF3Y606F and SLAMF3WT, respectively vs 8% of SLAMF3 positive cells in untreated HepG2 as checked by flow cytometry (Figure 3b, green events). Cells were challenged by HCVcc JFH1 strain for 72 hours and virus entry was evaluated by HCV E2 protein detection by flow cytometry using specific mAb. As presented in Figure 2b (pink events), after 72h post-infection (p.i.), 12% of HepG2 transfected by SLAMF3WT are positive for HCV E2. The intracellular HCV E2 positive cells decreased to 3 and 9 % of cells when endocytosis was blocked by ectopic transfection of SLAMF3Y470F SLAMF3Y606F (Figure 2 b). The objective of this study was to test the importance of SLAMF3 endocytosis in HCV SLAMF3 –dependent entry. Then, we used two forms of SLAMF3 in which endocytosis was partially abolished by site-directed mutagenesis to block the AP2 and Grb-2 binding sites on intracytoplasmic tail of SLAMF3. We show that rate of HCV E2 internalization is decreased by 25 and 75% in the presence of SLAMF3Y470F and SLAMF3Y606F, respectively. Our results confirm the importance of SLAMF3 in HCV entry that potentially uses SLAMF3 endocytosis and that the AP2 fixation on SLAMF3 is crucial for SLAMF3-dependent endocytosis of HCV.


References

  1. Marcq I., Nyga R., Cartier F., Ossart C., Lamotte L., Chatelain D., Ghamlouch H., Debuysscher V., Marolleau JP., Latour S.and Bouhlal H. SLAMF3 (CD229) expressed on hepatocytes inhibits hepatocarcinoma cell proliferation through ERK, JNK and mTOR pathways. Plos One, 2013
  2. Cartier F., Marcq I., Douam F., Ossart C., Regnier A., Debuysscher V., Lavillette D., Bouhlal H. The expression of the hepatocyte SLAMF3 (CD229) receptor enhances the hepatitis C virus infection. Plos One, 2014
  3. Del Valle, J.M., Engel, P., Martin, M. The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2). J Biol chem. (2003)
  4. Morra, M., Howie, D., Grande, M. S., Sayos, J., Wang, N., Wu, C., Engel, P., and Terhorst, C. (2001) Annu. Rev. Immunol. 19, 657–682
  5. Martın M, Del Valle JM, Saborit I and Engel P, Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229, J Immunol, 2005, 174: 5977–5986

 

 

HB fig1Figure 1: HCV entry model showing the main early and late receptors known to mediate HCV entry. GAGs; LDL-R low density receptors; Tetraspanin CD81, SR-BI scavenger receptor type I; CLDN1 tight junction proteins Claudin-1; OCLDN Occludin.

 

HB fig2Figure 2: Introduction of SLAMF3 into HepG2 cells. Cells from the HepG2 cell line were transfected with empty vector pBud (mock) or human SLAMF3 (HepG2+). The SLAMF3 expression level was assessed by flow cytometry and visualized by fluorescence microscopy; Results from one representative experiment are presented in (a) (unshaded histograms show the isotype-control and shaded histograms show SLAMF3 expression); (b) The expression level is presented as the mean fluorescence intensity (MFI) ratio (SLAMF3 MFI/Co MFI) (mean of three experiments; error bars: SD; *** p<0.005).

 

HB fig3Figure 3: HVC–SLAMF3-dependent entry use endocytosis machinery. a. diagram indicating the SLAMF3Y470F, SLAMF3Y606F and SLAMF3WT forms obtained by site-directed mutagenesis of SLAMF3; b. Cells (HepG2) were challenged with HCVcc JFH-1 strain for 3 hours and then cells were washed and cultured for 72h post-infection (p.i.). Cells were treated with Trypsin-EDTA buffer and stained at cell surface with anti-SLAMF3 mAb (1.9.25 isotype) for 20 min on ice. For intracellular HCV E2 staining, cells were fixed paraformaldehyde PFA 1% buffer and cell membranes permeabilized (BD Cytofix/Cytoperm™ Kits) before staining with anti-E2 mAb (BDI167 clone, Abcam) for 30 min. Then, cells were washed and analyzed using FACSAria cytometer (BD) and FlowJo software. The results are presented as dot plots from one of three representative experiments. The percent of HCV E2 and SLAMF3 positive cells is indicated at corresponding quadrants.

 

 

 

 

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