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

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

Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells

CAPN5 and CBLB have a scaffolding function in HCV infection.

<p>(A) CAPN5 (red) or CBLB (blue) knockout cells were complemented with sgRNA resistant CAPN5 (CAPN5; orange) / CBLB (CBLB; purple) or catalytically inactive CAPN5 (CAPN5 dead; light orange) / CBLB (CBLB dead; light purple), respectively. Infection of knockout and complemented cell lines with HCV genotype 2 reporter virus (upper panel). 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in knockout cells. Data from 3 independent experiments shown as mean +SEM. The protein expression level in knockout and complemented cell lines was analyzed by immunoblot (lower panel). Representative of 3 independent experiments. (B) HCV non-reporter infection in CAPN5 and CBLB knockout cells with and without complementation. Lunet N hCD81 cells with the indicated CRISPR and complementation construct were infected with non-reporter genotype 2 HCV (strain Jc1) and release of infectious particles measured at 72 h post infection by TCID50 assay. LOQ: limit of quantification. Shown are three (two for CAPN5 dead) independent experiments with technical duplicates each. (C) Schematic representation of CAPN5 and CBLB domain mutants. (D) The indicated truncated variants of CAPN5 and CBLB were overexpressed in Lunet N hCD81 cells and cells infected with <i>Renilla</i> reporter HCV. Infection was quantified 72 hpi by luciferase assay. PC: protease core; C2L: C2-like, TKB: tyrosine kinase binding; UBA: Ubiquitin associating; ctrl: empty vector control; scr: scrambled sgRNA. Bar graph shows mean + SD of one representative biological replicate (of four in total) with three technical replicates. Statistical analysis performed by ANOVA; ****p value<0.0001. (E) Model for the role of CAPN5 (red) and CBLB (blue) in HCV entry. Ub: ubiquitin, P: phosphate group. AP2: adaptor protein complex 2.</p

CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex.

<p>(A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.</p

Stratification of 33 CD81 receptor interactions in primary human hepatocytes.

<p><b>(</b>A) Schematic overview of the experimental setup used to define the CD81-interactome in primary human hepatocytes (PHH). (B) Immunoblot analysis of CD81- and IgG-IPs from PHH of two donors using an anti-CD81 antibody. Actin served as loading control. L = lysate, FT = flow through, E = eluate. (C) LFQ intensities of proteins in CD81- or IgG-IPs from PHH of two independent donors. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in PHH. (D) Scatter plot comparing intensity differences of proteins found in CD81- versus IgG-IPs in two donors of PHH. CD81 (green), SCARB1 (black), APOL2 (white) and CAPN5 (red) are highlighted. (E) Number of proteins found ≥ 10-fold enriched in the indicated co-IPs and membrane associated protein fraction. (F) 23 proteins found at least 4-fold enriched in CD81-IPs from PHH donor 1 and 2 and significantly enriched in co-IPs from Lunet N hCD81 and Lunet N hCD81HA. (G) 26 proteins identified in CD81-IPs from PHH donor 1 and 2 and in Lunet N hCD81 cells with high stringency (FDR < 0.004). Among the 26 proteins, 16 overlapped with the analysis in (F), resulting in a total of 33 stringent CD81 interactors in PHH and hepatoma cells. (H) Heat map showing protein abundance as median intensity (log₁₀) for the 33 hits and the CD81 bait in indicated co-IP samples. Red and blue colors indicate high or low intensity difference, respectively. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s012" target="_blank">S3 Table</a>.</p

A subset of CD81 interacting proteins is required for full HCV infectivity.

<p><b>(</b>A) Functional map of host factors interacting with the HCV receptor CD81. Functional clusters (boxes) and previously reported interactions (bold lines) of the identified CD81 binding partners and the HCV entry factors OCLN and CLDN1 are depicted. Yellow lines between genes of different clusters indicate high-confidence (>0.9) STRING interactions. Lower confidence (>0.35) STRING interactions are shown as red lines. Nine highest scoring additional nodes (indicated by asterisk) were included for follow up analysis. The full set of identified proteins is depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s003" target="_blank">S3A Fig</a>. (B) Experimental setup of the siRNA screen used to identify CD81 interactors important for HCV infection. (C) Human hepatoma cells were transfected with a pool of three siRNAs targeting the 42 CD81-interactors or with a scrambled non-targeting control (SiSel NC), followed by infection with a HCV luciferase reporter virus (JcR-2A). Infectivity was measured 48 hpi as luciferase activity and normalized for cell viability and plate effects. Knock down of four CD81-interactors significantly decreased HCV infection (p≤ 0.05; abs (z score) ≥ 2). Data from 3 biological replicates shown as mean +SEM. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s003" target="_blank">S3 Fig</a>.</p

CAPN5 and CBLB are HCV-specific, pan-genotypic host factors.

<p>(A) Schematic overview of the experimental setup used to infect CRISPR/Cas9 knockout cell lines with human coronavirus (hCoV) and vesicular stomatitis virus (VSV). (B) Infection with hCoV expressing a luciferase reporter. Infectivity quantified 24 hpi as luciferase activity (RLU, relative light units). (C) Infection with VSV encoding a GFP reporter. Infectivity analyzed 16 hpi by flow cytometry as mean fluorescence intensity (MFI). (D) Schematic overview of the experimental setup used to infect CRISPR/Cas9 knockout cell lines with HCVcc intergenotypic chimeras expressing the structural proteins of genotypes 1 and 3–7. (E-J) Infection of CRISPR/Cas9 knockout and parental cell lines with chimeric HCV expressing glycoproteins from genotype (GT) 1 and 3–7. Infectivity measured 72 hpi as luciferase activity and normalized to infection of Lunet N hCD81 cells transduced with non-targeting scrambled sgRNA. Data from three independent experiments shown as mean +SEM. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s006" target="_blank">S6 Fig</a>. Significance according to unpaired t-test (B, C, E-J) indicated by * (p≤ 0.05), ** (p≤ 0.01), *** (p≤ 0.001). Scr: scrambled sgRNA.</p

CAPN5 and CBLB support a postbinding step during HCV lipoviroparticle entry.

<p>(A) Schematic overview of the experimental setup used to analyze different steps of the HCV life cycle in the CRISPR/Cas9 knockout cell lines. (B) Infection of CAPN5 (red) and CBLB (blue) knockout and parental cell lines with HCV genotype 2 reporter virus. 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in cells transduced with a non-targeting scrambled sgRNA. CD81 knockout cells served as positive control. Data from 3 independent experiments shown as mean +SEM. (C) Flow cytometric surface staining of CD81, SCARB1, CLDN1 and OCLN in cells knocked out for CAPN5 (red) or CBLB (blue). Parental cells (black) served as positive control. Isotype control stainings or stainings with secondary antibody only (white) as negative controls. (D) Entry of lentiviral particles pseudotyped with glycoproteins from HCV GT1a (strain H77) or GT1b (strain Con1). Infectivity normalized to particles without envelope protein (negative control), to particles with VSV-G envelope (positive control) and to infection of cells transduced with non-targeting scrambled sgRNA. (E) Quantification of HCV fusion activity at the plasma membrane. Cells were pretreated with concanamycin A to inhibit endosomal acidification, cold-bound with HCV luciferase reporter virus (JcR-2A; 4°C, 2 h), shifted to 37°C (1 h) and washed with a pH 5 buffer to induce artificial plasma membrane fusion. A pH 7 buffer wash served to determine the background infection rate. 48 hpi infection rate was quantified as luciferase activity. Inh: flunarizine; scr: scrambled sgRNA (F) Immunofluorescence staining of cell lines electroporated with a HCV subgenomic replicon RNA (JFH1) at 48 hpt. Green: NS5A. Blue: DAPI. 10x magnification. (G) Cell lines were electroporated with wildtype HCV subgenomic replicon RNA (JFH1) or a polymerase active site mutant JFH1-ΔGDD (dotted lines), both encoding a luciferase reporter. Replication quantified as luciferase activity at the indicated time point post electroporation. Results normalized to the 4 h time point to account for electroporation efficiency. Data from at least three independent experiments shown as representative results (C, F) or as mean ± SEM (B, D, E, G). Significance according to unpaired t-test (B, E) or to MANOVA (G) indicated by * (p≤ 0.05), ** (p≤ 0.01), *** (p≤ 0.001). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007111#ppat.1007111.s006" target="_blank">S6 Fig</a>.</p

Proteins in the human liver cell CD81 complex.

<p>Proteins in the human liver cell CD81 complex.</p

Sequence of sgRNAs and primers used for CRISPR/Cas9 knockout cell generation and evaluation.

<p>Sequence of sgRNAs and primers used for CRISPR/Cas9 knockout cell generation and evaluation.</p