The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection

<div><p>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.</p></div

Recombinant viral E2 (rE2) interacts with SLAMF3.

<p>(A) Mock and Huh-7+ cells were fixed (in 4% PFA for 10 min) prior to incubation with rE2 for 1 h at 4°C. To measure binding inhibition, cells were incubated with anti-CD81 (JS-81), anti-SLAMF3 (3H) or isotypes for 30 min prior to incubation with rE2 and detection of its binding with anti-E2 antibody by flow cytometry (the mean of two experiments; error bars: SD); (B) Extracted proteins from Huh-7+ and COS-7 were incubated with coated rE2 on an ELISA plate before extensive washing and specific detection of bound CD81 (JS-81) and SLAMF3 (3H) proteins. The optical density (OD) was read at 580 nm and is presented as the mean of two experiments; error bars: SD; (C) Recombinant soluble HCV E2 (rE2) and bovine serum albumin (BSA, used as a control) were coated on nitrocellulose membranes (at the indicated concentrations) prior to incubation with 3/11 E2-specific antibody; (D) rE2 and BSA (5 µg) were coated on nitrocellulose membranes and then incubated with proteins from Huh-7+ cells. Spots were detected using anti-E2 (positive control), anti-SLAMF3 (K12 and HLy9.1.25) and anti-CD81 (JS-81) antibodies; (E) CD81 and SLAMF3 bound to rE2 used at the indicated concentrations were incubated with anti-CD81 (JS-81) and anti-SLAMF3 (HLy9.1.25) antibodies; (F) proteins from native COS-7 cells (top; mock) or SLAMF3-over-expressing COS-7 cells (bottom) were incubated with rE2 and BSA coated on nitrocellulose membranes prior to CD81 and SLAMF3 detection with specific antibodies. One representative experiment from three independent experiments is presented in A, D, E and F.</p

SLAMF3 over-expression increases hepatocyte permissiveness to HCV.

<p>Huh-7 cells were transfected with empty vector pBud (mock) or human SLAMF3 (to yield Huh-7+ cells) and incubated with HCVcc. (A) SLAMF3 expression was analyzed by flow cytometry (HLy9.1.25 clone) and (B) visualized by confocal microscopy in one out of three experiment. Unshaded histograms show the isotype-matched control (Co) and shaded histograms show SLAMF3 expression). (C, D, E) Infection was assessed at 4 and 72 h p.i. as the number of FFUs and (F and G) by flow cytometry analysis; (C) intracellular E2 (stained red) were measured at 4 h and 72 h p.i. Green staining corresponds to the SLAMF3 expression. One of three representatives independent experiments is shown. (D) The HCV permissiveness of Huh-7 mock and Huh-7+ cells was evaluated in terms of the number of FFUs based on intracellular E2 viral protein staining and (E) fluorescent microscopy analysis (mean of five experiments; error bars: SD; *p<0.05); Infection evaluation by flow cytometry analysis measuring the intracellular viral E2. (F) Infected cells (72 p.i.) were stained for surface expression of SLAMF3 (HLy9.1.25 clone) and permeabilized for intracellular E2 staining. The percentage of positive cells in each cell population is indicated in the corresponding quadrant (one of three representative experiments is shown) and (G) as a mean of three experiments (error bars: SD; *p<0.05).</p

SLAMF3 over-expression increases the HCV infectivity.

<p>Mock and Huh7+ cells were challenged with HCVcc (A, top panel). (A) Supernatants were harvested at 72 h p.i., released viruses quantified by QPCR and equal viral loads from both supernatants were used to infect native Huh-7 cells (A bottom panel). (B) Viral replication was assessed by intracellular viral E2 protein expression measurement by flow cytometry and quantitation of released viral RNA at 72 h p.i. (mean of three independent experiments; error bars: SD *p<0.05, **p<0.01).</p

Hepatocyte SLAMF3-derived peptides inhibit HCV infection.

<p>For peptide competition assays, peptides derived from the first (P1D1 and P2D1), second (P1D2) and third (P1D3) extracellular domains of SLAMF3 and irrelevant peptide (i.p.) used at 0.25 µM were incubated with HCVcc prior to Huh-7 cells infection. (A) The diagram shows SLAMF3's topology and the position of SLAMF3 peptides used to prevent HCV attachment and infection; (B) the inhibitory effect was evaluated by quantification of intracellular viral RNA in infected Huh-7 cells at 72 h p.i. (mean of three experiments; error bars: SD; *p<0.05; **p<0.01, ns: non significant) and (C) in primary human hepatocytes (PHHs) presented as mean of one experiment; (D) Huh7 cells were challenged with HCVcc H77/JFH-1 and the inhibitory effect of SLAMF3-derived peptides was evaluated at 72 h p.i. and presented as FFU values (mean of three experiments; error bars: SD; *p<0.05); (E) The inhibitory effect of SLAMF3-derived peptides was tested on different pseudoparticles harboring envelope glycoproteins derived from HCV genotypes 1a-H77; 2a-HC-J6/CF; 3a-UKN3A1.28 and 4-UKN4.21.16 or from VSV (used as a control to normalize infection assays), (mean of three independent experiments; error bars: SD; *p<0.05, **p<0.01, ns, non-significant).</p

SLAMF3 blockade in human hepatocytes is associated with lower susceptibility to HCV.

<p>(A) SLAMF3 was stained in primary human hepatocytes (PHHs) and cells from the Huh-7 human hepatoma cell line with a specific antibody (HLy9.1.25 clone; grey) and an isotype-matched control (empty). One of four independent experiments is shown. Huh-7 cells were transfected with scrambled control (sc) siRNA or three specific siRNAs (#1, #2 and #3) targeting SLAMF3, prior to infection with HCVcc; siRNA efficiency was checked by quantifying SLAMF3 mRNA (B) and the CD81 expression level (C) by flow cytometry analysis at 48 h post-transfection. Results are presented as the mean ±SD (n = 3). Intracellular viral RNA was quantified at 72 h p.i. (D) and the infection was measured at 72 h p.i. by focus-forming units FFUs counting (E) (as inhibition percent; mean of three independent experiments; error bars: SD. **p<0.01);</p

Antibodies against hepatocyte SLAMF3 reduce HCV infection.

<p>(A) Cells from the Huh-7 line were treated with anti-CD81 (JS-81) and/or anti-SLAMF3 (3H, ZY and HLy9.1.25 clones) mAbs prior to HCVcc infection (evaluated by quantification of intracellular vial RNA at 72 p.i.). (B) Cultured primary healthy human hepatocytes PHH were treated with anti-CD81 (JS-81) and/or anti-SLAMF3 (3H) mAbs prior to HCVcc infection (evaluated by quantification of intracellular viral RNA at 72 h p.i.). (Ct) refers to an isotype-matched control (in A and B), all neutralizing antibodies were used at 10 µg/ml; (C) The inhibitory effect of the 3H mAb on the number of FFUs after Huh-7 infection was checked at 72 h p.i. (mean of three independent experiments; error bars, SD * p<0.05, **p<0.01, ***p<0.005, ns: non-significant).</p

Analysis of HCV particles secreted from cells that over-express ABCA1.

<p>Physical properties of the nascent virus particles produced in cells stimulated or not with GW3965 were analysed by centrifugation in iodixanol gradient. Huh7.5 cells were pre-incubated with solvent (panel A) or 1 µM GW3965 (panel B) and the drug was maintained until 72 h post-infection when cell supernatants were collected, concentrated and subjected to gradient centrifugation. HCV RNA in gradient fractions was quantified by qRT-PCR and core antigen, ApoB and ApoE by ELISA assays.</p

Up-regulation of ABCA1 inhibits HCV cell entry.

<p>The effect of GW3965 on the HCV cell cycle was analysed by adding the drug at different time points. A flow-chart is depicted in the upper panel of the graph. RNA in Huh7.5 cells infected in the presence of DMSO is shown in (a); that in cells pre-treated for 24 h with 1 µM GW3965 and infected in the presence of the drug are shown in (b), (c) and (d); results for cells treated with GW3965 during virus inoculation without pre-treatment are shown in (e); those of assays where the drug was added at 2 h, 4 h, or 6 h post-infection are presented in (f), (g) and (h) respectively. For each experiment cells were incubated for the indicated time period after infection (IV). The efficiency of infection was expressed as intracellular HCV RNA measured by qRT-PCR as a per cent of the control (a).</p

GW3965 treatment up-regulates ABCA1 expression and its cholesterol efflux function.

<p>(A) Cell toxicity of GW3965. Huh7.5 cells were cultured in the presence of indicated concentrations of the drug for 24 h. The luminescent signal is expressed in luminescence units (RLU). (B) Up-regulation of ABCA1 mRNA expression by GW3695 treatment. Huh7.5 cells were treated for 24 h with 1 µM GW3695 or drug solvent (DMSO). Then ABCA1 mRNA was determined by qRT-PCR. (C) ABCA1 protein production in drug-stimulated Huh7.5 cells. Cells were treated for 24 h with 1 µM GW3965 and analysed by Western blot (shown in the insert). Protein content in the ABCA1 band (220 kDA) in GW3965-(GW), and DMSO-(solv) treated cells was quantified relative to the calnexin band using the Odyssey Infrared Imaging System. (D) GW3965 stimulation promotes ABCA1-mediated cholesterol efflux to ApoA1. Huh7.5 cells were labelled with [³H] cholesterol then incubated with GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed by comparing cell-associated and free radioactivity. (E) Kinetics of ABCA1 gene expression following stimulation of cells with GW3965. Huh7.5 cells were treated with 1 µM GW3965 for the indicated time and ABCA1 mRNA was determined by qRT-PCR. Results were expressed as relative values compared to ABCA1 expression in cells treated with drug solvent. (F) Kinetics of cholesterol efflux in cells stimulated with GW3965. Huh7.5 cells were labelled with [³H] cholesterol for 24 h, and incubated for an additional 16 h with 1 μM GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed in the presence of ApoA1 and either GW3965 or solvent for the indicated period of time.</p