Fig 1.
ADAM10 is highly enriched in CD81 affinity purifications from hepatoma cells and primary human hepatocytes.
(A) Experimental design of affinity enrichment mass spectrometry for the identification of the CD81 protein complex in human hepatoma cell lines (IntAct entry https://www.ebi.ac.uk/intact/search?query=IM-25678). (B) Results of two-sample t-tests comparing protein label free quantification (LFQ) intensities of CD81 co-IPS from Lunet N hCD81 cells (CD81) with those of Lunet N (CTRL) displayed as a volcano plot. (C) Same as in (B), comparing protein LFQ intensities of HA co-IPS from Lunet N hCD81-HA (CD81) with those of Lunet N (CTRL). (D) Ranked protein abundances in whole cell proteomes of human hepatoma cells. (E) Protein enrichment in CD81 co-IPs from primary human hepatocytes (PHH) of two independent donors. Isotype control antibodies were used as control (CTRL). (F) Transcript expression of ADAM10, HCV entry factors, receptors transactivated by ADAM10 (EGFR, ERBB2, ERBB3, TNFRSF1A) and negative controls (CD8A, CLEC4M) across cells in healthy human liver tissue from nine donors using the cell type annotation from Aizarani et al. [57]. The size of the dots represents the percentage of transcript positive cells per cell type with at least one detected transcript for the respective gene, and the colour the average expression in log2 scale. Mass spectrometry datasets show median values of four independent experiments for the hepatoma cells. Proteomics data derived from and described in (Bruening et al. 2018) [16].
Fig 2.
ADAM10 is a host factor for hepatitis C virus.
(A,B) ADAM10 (A) and CD81 (B) surface expression levels measured by antibody staining and flow cytometry on Huh-7.5-Fluc cells treated with a pool of 3 siRNAs targeting ADAM10, CD81, or a non-targeting siRNA control. Mean fluorescence intensity (MFI) values normalized to control siRNA are shown. (C,D) ADAM10 (C) and CD81 (D) protein expression levels determined by immunoblot of lysates from Huh7.5-Fluc cells treated as in (A). (E) Huh-7.5-FLuc cells were treated as in (A) and (B) for 48 h and infected with a Jc1-based renilla luciferase reporter virus (JcR2A). Infectivity was quantified 72 hpi as renilla luciferase activity. Infectivity values are normalized to non-targeting siRNA control. (F) Cell viability of cells treated as in (E) was assessed by MTT assay. (G). ADAM10 surface expression in cells treated with the ADAM10 inhibitor GI254023X or DMSO was measured as in (A). (H) HCV infection of cells pre-treated with GI254023X or SR-BI inhibitor ITX5061 for 16h. Virus inoculum was supplemented with inhibitors, removed 4 hpi and replaced with medium supplemented with inhibitors. Infectivity was measured and values were normalized as in (E). (I) Cell viability upon inhibitor treatment as in (H) was assessed by MTT assay and values were normalized to DMSO control. (J) Huh-7.5-FLuc cells were treated with a sgRNA targeting ADAM10 exon 11 and a non-targeting sgRNA control. ADAM10 expression was measured as in (A). (K) ADAM10 KO and control cells were infected as in (E). Infectivity was measured as described in E and values were normalized to non-targeting control. (L) Huh-7.5-Fluc cells were transduced with lentiviruses encoding ADAM10 (ADAM10 OE) or control lentiviruses (EV). Surface expression levels of ADAM10 overexpressing cells were quantified by antibody staining and flow cytometry. (M) Huh-7.5-Fluc cells were transduced as in (L). 72 h post transduction, cells were infected with JcR2a. 72 h post infection, infection was quantified as in (E). (N) ADAM10 KO and control cells were transduced as in (L). Cells were then infected with JcR2a 72 h post transduction. Infectivity was measured 72 hpi as in (E). Data show the mean +/- SD of three biological replicates. One-way ANOVA with Dunnett´s multiple comparison test (E and H). Paired t-test (K and M). * P < 0.05; ** P < 0.01; *** P ≤ 0.001.
Fig 3.
ADAM10 promotes HCV lipoviroparticle entry and cell-to-cell spread but is dispensable for HCV replication and HCV pseudoparticle entry.
(A) Scheme of the generation of E1/E2 pseudotyped lentivirus. Pseudoparticles encode a firefly luciferase for quantification of transduction efficiency. (B) Huh-7.5 cells treated for 48 h with pools of 3 siRNAs targeting ADAM10 or CD81 or a non-targeting control (CTRL) were transduced with lentiviruses pseudotyped with HCV E1/E2 glycoproteins. 72 h post transduction, transduction efficiency was measured as firefly luciferase activity. Values represented relative to non-targeting control (CTRL). (C) Huh-7.5 cells were treated with the ADAM10 inhibitor GI254023X and the SR-BI inhibitor ITX 5061 for 16 h and transduced with the lentiviruses described in (A). Infectivity was measured 72 h post transduction as in (B). (D,E) Huh-7.5-Fluc cells were electroporated with a wildtype (D) or replication deficient ΔGDD mutant (E) HCV subgenomic replicon (SGR) encoding firefly luciferase and treated with ADAM10 inhibitor GI254023X or the NS3/4A inhibitor telaprevir during and after electroporation. HCV subgenome replication was assessed as firefly luciferase activity. Data show the fold change over 4 h luciferase activity values (F) ADAM10 surface expression was measured by antibody staining and flow cytometry at the indicated time points post 12 h inhibitor treatment and washout. Mean fluorescence intensity (MFI) values are normalized to DMSO at each time point. (G) Scheme of the ADAM10 inhibitor time of addition assay. Huh-7.5-Fluc cells were treated with ADAM10 inhibitor GI254023X or DMSO prior, during or after HCV virus inoculation. (H) HCV infection of cells treated with inhibitor as shown in (G). Infectivity was measured by renilla luciferase assay at 72 hpi. Infectivity values are normalized to DMSO controls for each time point. (I) Scheme showing the HCV cell-to-cell spread assay. Huh-7.5-Fluc cells were infected with GFP-tagged HCV (MOI 0.1). 7 dpi, Huh-7.5-Fluc GFP+ cells were mixed with uninfected Huh-7.5-Fluc cells pretreated with the ADAM10 inhibitor GI254023X, the matrix metalloprotease inhibitor BB-94, or DMSO at a 1:5 ratio. 4 h later, cells were overlayed with Avicel-containing medium supplemented with inhibitors or DMSO. Infectivity was measured 72 hpi using flow cytometry. (J) Percentage of GFP positive cells 7 dpi. (K) Cell-to-cell spread of HCV measured as percentage of GFP positive cells in the conditions described in (I) was normalized to DMSO controls. Data shows the mean +/- SD of three biological replicates. Two- way ANOVA with Dunnett´s multiple comparison test (D). Unpaired t-test (H). One-way ANOVA with Dunnett´s multiple comparison test (K). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Fig 4.
ADAM10 sheddase activity promotes HCV infection.
(A) Surface expression of HCV entry factors on Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO for 72 h. Values represent mean and SD of the mean fluorescence intensity of three independent experiments and are presented as percentage of DMSO control values. (B) Cell-to-cell spread of HCV in Huh-7.5-Fluc cells treated with the matrix metalloprotease inhibitor BB-94 was quantified as in Fig 3K. (C) ADAM10 surface expression upon BB-94 or DMSO treatment quantified as in (A). (D) HCV infection of Huh-7.5-Fluc cells treated with non-targeting siRNA control or pools of 3 siRNAs targeting ADAM17 or CD81. 48 h post siRNA transfection, cells were infected with JcR2a for 72 h. Infection levels were measured by renilla luciferase assay and normalized to the non-targeting control. (E) Huh-7.5-Fluc cells were treated as in (D). 48 h post siRNA transfection, ADAM17 surface expression levels were analyzed by antibody staining and flow cytometry (F) Scheme of receptors and downstream kinases activated by ADAM10 sheddase activity. (G) Representative histogram showing increased ERK1/2 phosphorylation upon EGF stimulation in DMSO-treated, serum-starved cells. (H) ERK1/2 phosphorylation in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h or left in medium containing serum and stimulated with 1 μg/mL EGF for 15 min. Values represent mean and SD of the mean fluorescence intensity of four independent experiments and presented as percentage of DMSO control values. (I,J) pERK1/2 (I) and total ERK1/2 (J) protein levels analyzed by immunoblot from Huh-7.5-Fluc cell lysates after serum starvation and EGF stimulation for 15 min as in (G). (K,L) pEGFR levels measured by flow cytometry (K) or immunoblot (L) in Huh-7.5-Fluc cells treated with ADAM10 inhibitor GI254023X or DMSO for 48 h, serum starved for 12 h and stimulated with EGF for 15 min. Mean fluorescence intensity analysis as in (A). (M) Rescue of ADAM10 KO cell infection phenotype by pretreatment 16 h prior to and during HCV infection with the ADAM10 substrates TNF-α, EGF, or E-cadherin. Infection measured as renilla luciferase activity as in (D) and values normalized to the respective scrambled guide RNA control. (N) Relative cell number of ADAM10 KO and control cells upon treatment with ADAM10 substrates as in (M). Values normalized to scrambled guide RNA control cells. Data show the mean +/- SD of three biological replicates unless stated otherwise. One-way ANOVA with Dunnett´s multiple comparison test (B, D). Paired t-test (, E, H, M). * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 5.
ADAM10 is dispensable for infection with alphaviruses and coronaviruses.
(A) CHIKV-GFP (ECSA LR2006) infection of Huh-7.5-Fluc cells treated with the ADAM10 inhibitor GI254023X or DMSO (MOI 1). Infection levels at 24 h were measured by quantification of GFP signal and normalization to DMSO control. (B) VEEV-GFP (TC-83) infection (MOI 0.1) of Huh-7.5-Fluc cells treated with ADAM10 inhibitor or solvent control as in (A). (C) Renilla luciferase reporter hCoV-229E infection of Huh-7.5-Fluc cells treated as in (A). 72 h post infection, renilla luciferase activity was determined as a measure of infection. Data show the mean +/- SD of three biological replicates.
Fig 6.
Effect of ADAM10 inhibition on HCV infection of primary human hepatocytes.
(A) ADAM10 surface expression on primary human hepatocytes (PHH) upon ADAM10 inhibitor GI254023X treatment in presence or absence of JAK-STAT inhibitor ruxolitinib at 96 h post inhibitor treatment. (B) Quantification of ADAM10 surface staining as in (A) for PHH from three independent donors measured as mean fluorescence intensity (MFI). (C) Effect of ADAM10 inhibition on PHH infection with HCV upon pre-treatment with the Jak-STAT inhibitor ruxolitinib with and without ADAM10 inhibitor GI254023X for 24h. Cells were then infected with cell-culture adapted HCV P100 (supplemented with ruxolitinib and GI254023X as indicated) at MOI 1. Virus inoculum was removed 5h post infection and replaced with medium containing ruxolitinib and GI254023X as indicated. Virus supernatants were collected 24, 48 and 72 hpi and titrated on Huh-7.5 cells by TCID50. (C). Data show the mean +/- SEM of three biological replicates, i.e. PHH from three independent donors (B,C) or a representative experiment (A). Two-way ANOVA with Sidak´s multiple comparison test (ns = non-significant).