CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells

Abstract

Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

Keywords: CD81; HCV; chimeras; cholesterol-binding residue; hepatitis C virus; receptor; susceptibility-determining domains; tetraspanin; transmembrane domain four.

Figure 1

Expression of hCD81 chimeras on CHO745 cells. (A) Schematic representation of the hCD81 chimeras used in this study; (B) Amino acid sequence identities calculated by Clustal W and domain structure of the chimeras; (C) Flow cytometric analysis of surface expression on CHO745 cells. Cells were transduced with the indicated hCD81 chimeras, full-length hCD81 (wt) or empty vector (Ctrl) and selected with blasticidin. CD81 surface expression was determined by staining with an antibody directed against the hCD81 LEL (clone JS-81). The histograms show cells stained with anti-hCD81, an isotype control antibody or cells left unstained. Vertical lines indicate gating for CD81 positive cells; (D) Quantitation of hCD81 surface staining depicted as percentage of positive cells or (E) as mean fluorescence intensity (MFI). Mean values + SD of three independent biological replicates are shown in (D,E). The histograms show one out of three representative experiments with 30,000 cells per measurement.

Figure 2

HCV E2 differentially binds to CHO745 cells expressing hCD81 backbone chimeras. (A) Binding of Alexa-647 labeled soluble E2 ectodomains (eE2) of the HCV genotypes 1a (strain H77) and 2a (strain J6) with and without the hypervariable region (HVR) to CHO745 cells expressing the indicated hCD81 chimera, full-length protein (wt) or empty vector control (Ctrl). Each histogram shows fluorescence intensities of untreated cells (grey) and cells after Alexa-647 eE2 binding (white); (B–E) Quantitation of eE2 binding to CHO745 cells expressed as MFI of Alexa-647. CHO745 cell expressing SR-BI served as additional positive control. Data show one representative experiment of three independent biological replicates.

Figure 3

Generation and characterization of hCD81 chimera expressing human hepatoma cells. (A,B) Immunoblot for hCD81 LEL (A) and HA tag (B) in lysates from Lunet N#3 human hepatoma cells stably transduced with the indicated tetraspanin construct. GAPDH served as loading control and lysates from Huh-7.5 cells with endogenous hCD81 as positive control; (C–E) Cell surface staining of hCD81 on transduced Lunet N#3 cells as described in Figure 1C–E. Immunoblots and histograms are representative of at least three independent experiments and histograms show 30,000 cells per measurement. Mean + SD of three independent experiments shown.

Figure 4

Subcellular distribution analysis of the five surface expressed hCD81 chimeras and full length hCD81 in Lunet N#3 and Huh-7.5 cells. (A) Confocal microscopy of hCD81 LEL stained Lunet N#3 cells expressing the indicated tetraspanin chimeras. Cells were permeabilized and stained with anti-hCD81 antibody (clone JS-81, green). Nuclei were stained with DAPI (blue). Huh-7.5 cells served as positive control, scale bars = 20 µm (B) Confocal microscopy of Lunet N#3 and Huh-7.5 cells stained with anti-HA antibody (red), anti-p230 antibody (green) as Golgi marker and DAPI (blue). Representative images; scale bars = 20 µm. (C) Semi-quantitative analysis of hCD81 chimera subcellular localization. +, expression observed; −, expression not observed; N.A., data not available.

Figure 5

The hCD81 backbone influences HCV pseudoparticle infectivity. (A) Chimera expressing Lunet N#3 cells and Huh-7.5 cells were transduced with lentiviral pseudoparticles encoding Firefly luciferase and displaying the glycoproteins of HCV (strain H77) or VSV. Pseudoparticles without glycoproteins (no Env) served as negative control. Pseudoparticle infectivity was measured at 72 hpt by quantitation of luciferase activity; (B) Infectivity of GFP encoding lentiviral pseudoparticles displaying HCV (H77) glycoproteins, VSV glycoproteins, or no glycoproteins in chimera expressing Lunet N#3 cells and Huh-7.5 cells. At 72 hpt cells were counterstained with anti-hCD81 antibody, and infectivity of lentiviral GFP pseudoparticles in the hCD81 LEL positive subpopulations quantified. n.d., not detected, i.e., CD81 positive cells below 1000 counts. All experiments were performed at least thrice and are represented as mean + SD of three independent biological replicates (A) or technical triplicates (B).

Figure 6

The hCD81 backbone determines HCV infectivity, but not human coronavirus and VSV infectivity. (A) Infectivity of a luciferase reporter HCV strain (Jc-R2A) in Lunet N#3 cells expressing hCD81 chimeras and in Huh-7.5 cells. Cells were infected for 72 h at an MOI of 0.5 and infectivity quantified as luciferase activity in cell lysates; (B) Infectivity of non-reporter full-length HCV (Jc1) in hCD81 chimera expressing cells. Viral titers at 72 hpi were determined by limiting dilution on Huh-7.5 cells and immunocytochemical staining against the viral NS3 protein; (C) Replication assay using an HCV subgenome (JFH-1). Cell lines were electroporated with JFH-1 subgenomic RNA and intracellular viral RNA was quantified by real time PCR at the indicated timepoint; (D) Human coronavirus (strain 229E) infectivity determined at 48 hpi using Renilla luciferase reporter virus; (E) VSV infectivity determined at 16 hpi using GFP reporter virus; (F) HCV (JcR2A) infectivity through artificially induced plasma membrane fusion. Virus was bound in the presence of a V-type ATPase inhibitor and fusion induced at the plasma membrane by a low pH wash. The fusion inhibitor flunarizine (Inh.) served as control. Data shown as mean + SD (SEM in (F)) of three to five independent biological replicates each performed in technical triplicates.

Figure 7

HCV genotypes similarly depend on the hCD81 backbone. (A) Lunet N#3 cells expressing the hCD81 chimeras and Huh-7.5 cells were infected with intergenotypic chimeric viruses encoding Renilla luciferase reporter. At 72 hpi, cell lysates were analyzed for luciferase activity as a measure of HCV infectivity. Graphs show three independent biological replicates (mean of technical triplicates each); (B) Influence of CD81 variation on HCV susceptibility of Lunet N#3 cells. The HCV genotype 2a susceptibility (normalized to hCD81 expressing Lunet N#3 cells) is plotted on the y axis and the identity of the respective CD81 chimera to hCD81 on the x axis. Exponential growth regression analysis with all negative values excluded from analysis; (C) Sensitivity of HCV genotypes to CD81 backbone variation plotted as rate constant (k) of the exponential growth regression analysis for HCV genotype 1–7 intergenotypic chimeras. Statistical significance in (C) tested by ANOVA with no significant differences observed.

Figure 8

Transmembrane domain four of dmTSP90F and cdTSP9 critically differ from hCD81. (A,B) Scheme of the structural domains of hCD81 and amino acid sequence divergence scan of hCD81 chimeras with HCV entry supporting (A) or resistant (B) backbones; (C) Amino acid sequence alignment of the transmembrane domain four (TM4) and the N-terminus of the tetraspanin chimeras used in this study. The cholesterol binding residue E219 is highlighted in light blue. A conserved palmitoylation site and an internalization motif are highlighted in black; (D) Structure of full length hCD81 with bound cholesterol (light red, ball and stick) and the E219 residue (blue, ball and stick). The boundaries of the plasma membrane are indicated by horizontal lines. (PDB: 5TCX) [19]. CD81 amino acids are colored according to the side chain hydrophobicity (polar: blue, nonpolar: red). Disulfide bonds in LEL highlighted in orange. The insert shows the zoom into the E219–cholesterol interface formed by hydrogen bonds between the E219 carboxyl group and the cholesterol hydroxyl group (oxygen atoms labelled in red, nitrogen in blue). (E) Cell surface staining of hCD81 on TM4 mutant transduced Lunet N#3 cells as described in Figure 1D. (F) Infectivity of luciferase reporter HCV strain (Jc-R2A) in hCD81 TM4 mutant expressing Lunet N#3 cells as described in Figure 6A. Data shown as mean + SEM of four independent biological replicates each performed in technical triplicates. ** p ≤ 0.01, *** p ≤ 0.001.

Figure 8

Transmembrane domain four of dmTSP90F and cdTSP9 critically differ from hCD81. (A,B) Scheme of the structural domains of hCD81 and amino acid sequence divergence scan of hCD81 chimeras with HCV entry supporting (A) or resistant (B) backbones; (C) Amino acid sequence alignment of the transmembrane domain four (TM4) and the N-terminus of the tetraspanin chimeras used in this study. The cholesterol binding residue E219 is highlighted in light blue. A conserved palmitoylation site and an internalization motif are highlighted in black; (D) Structure of full length hCD81 with bound cholesterol (light red, ball and stick) and the E219 residue (blue, ball and stick). The boundaries of the plasma membrane are indicated by horizontal lines. (PDB: 5TCX) [19]. CD81 amino acids are colored according to the side chain hydrophobicity (polar: blue, nonpolar: red). Disulfide bonds in LEL highlighted in orange. The insert shows the zoom into the E219–cholesterol interface formed by hydrogen bonds between the E219 carboxyl group and the cholesterol hydroxyl group (oxygen atoms labelled in red, nitrogen in blue). (E) Cell surface staining of hCD81 on TM4 mutant transduced Lunet N#3 cells as described in Figure 1D. (F) Infectivity of luciferase reporter HCV strain (Jc-R2A) in hCD81 TM4 mutant expressing Lunet N#3 cells as described in Figure 6A. Data shown as mean + SEM of four independent biological replicates each performed in technical triplicates. ** p ≤ 0.01, *** p ≤ 0.001.