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. 2011 May;17(5):589-95.
doi: 10.1038/nm.2341. Epub 2011 Apr 24.

EGFR and EphA2 Are Host Factors for Hepatitis C Virus Entry and Possible Targets for Antiviral Therapy

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Free PMC article

EGFR and EphA2 Are Host Factors for Hepatitis C Virus Entry and Possible Targets for Antiviral Therapy

Joachim Lupberger et al. Nat Med. .
Free PMC article

Abstract

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests. Inserm and the University of Strasbourg have filed a patent application on–Host cell kinases as targets for antiviral therapy against HCV infection.

Figures

Fig. 1
Fig. 1. EGFR is a co-factor for HCV entry
(a,b) Silencing EGFR expression in HCV-permissive cells. (a) EGFR mRNA (qRT-PCR analysis) and (b) protein expression (Western blot) in Huh7.5.1 cells transfected with EGFR-specific individual siRNAs (si1–4). Silencing of CD81 mRNA expression by CD81-specific siRNA served as control. EGFR mRNA (relative to GAPDH mRNA) and protein expression compared to cells transfected with control siRNA (siCTRL) is shown. (c,d) Inhibition of HCV infection and entry in cells with silenced EGFR expression. (c) HCVcc infection in Huh7.5.1 cells transfected with individual siRNAs shown in panels a,b. siCTRL and CD81-specific siRNA served as internal controls. Data are expressed as percentage HCVcc infection relative to siCTRL-transfected cells. (d) Entry of HCVpp containing envelope glycoproteins of various isolates14],39] in Huh7.5.1 cells transfected with si4. VSV and measles virus pp entry or cells transfected with CD81 siRNA served as controls. Data are expressed as percentage pp entry relative to siCTRL-transfected cells. (e,f) Rescue of HCV entry in cells with silenced EGFR expression by exogenous EGFR. (e) HCVpp entry and EGFR protein expression in Huh7.5.1 cells co-transfected with EGFR-specific individual si3 and a cDNA encoding for RNAi-resistant EGFR (pEGFR-WT)40]. (f) HCVpp entry and EGFR protein expression in PHH co-transduced with lentiviruses expressing shEGFR and wild-type EGFR cDNA (EGFR-WT)40]. Protein expression was quantified using Image Quant analysis of Western blots. Data are expressed as percentage HCVpp entry relative to CTRL cells or as percentage EGFR expression normalized for β-actin expression. *** P<0.0005.
Fig. 2
Fig. 2. Inhibition of EGFR activation by kinase inhibitors reduces HCV entry and infection
(a) Effect of Erlotinib on HCV entry and infection in Huh7.5.1 cells. HCVcc (Luc-Jc1; J6-JFH1) infection and HCVpp (J6) entry in Huh7.5.1 cells pre-incubated with indicated concentrations of Erlotinib are shown. Data are expressed as percentage HCVcc infection or HCVpp entry relative to solvent-treated control cells (means ± SEM). (b) Effect of Erlotinib on HCV replication. Northern blot analysis of HCV RNA and GAPDH mRNA in Huh7.5 cells electroporated with RNA from subgenomic HCV JFH1 replicon and incubated with solvent CTRL, HCV protease inhibitor BILN-2061 or Erlotinib (ERL) is shown. Analysis of HCV RNA in cells transfected with replication incompetent HCV RNA (GND, Δ) served as negative control. (c) Effect of Erlotinib on HCVpp and MLVpp entry in HepG2-CD81 cells. Pseudovirus entry into non-polarized and polarized HepG2-CD81 cells (generated as described15]) pre-incubated with Erlotinib (10 μM) is shown. (d) Effect of Erlotinib on HCVpp entry into PHH. HCVpp entry in PHH pre-incubated with Erlotinib is shown relative to entry into solvent-treated control cells. IC50 value is expressed as median of three independent experiments ± standard error of the median. (e,f) Effect of PKIs on HCV entry and infection in PHH and Huh7.5.1 cells. (e) HCVpp entry into PHH and (f) HCVcc infection in Huh7.5.1 pre-incubated with 1 μM Erlotinib (ERL), Gefitinib (GEF), Lapatinib (LAP), Blebbistatin (BLEB) or Wortmannin (WORT) is shown. Cell viability was assessed using MTT assay.
Fig. 3
Fig. 3. Modulation of HCV entry by EGFR ligands and an EGFR-specific antibody
(a) Modulation of EGFR phosphorylation by EGF, Erlotinib and EGFR-specific antibody. EGFR activation was assessed in PHH incubated with the indicated compounds using the Human Phospho-RTK Array Kit. Phospho-tyrosine (P-Tyr) and phosphorylation of an unrelated kinase (MERTK) served as internal positive and negative controls. (b,c) Effect of EGFR ligands on HCVpp entry. HCVpp entry (HCV-J) into serum-starved Huh7.5.1, polarized HepG2-CD81 and PHH in the presence of EGF (b) and TGF-α (c) is shown. (d) Reversion of EGF mediated-enhancement of HCVpp entry by Erlotinib. HCVpp entry into Huh7.5.1, polarized HepG2-CD81 and PHH incubated with EGF or EGF and Erlotinib is shown. (e) Flow cytometric analysis of non-permeabilized PHH binding EGFR-specific or control monoclonal antibody (mAb). (f) Inhibition of HCV entry by EGFR-specific mAb. HCVpp entry into PHH pre-incubated with EGFR-specific or control mAb is shown. Viability of cells was assessed using MTT assay. IC50 value is expressed as median of three independent experiments ± standard error of the median. (g) Reversion of EGF-induced enhancement of HCV entry by an EGFR-specific antibody. HCVpp entry into PHH pre-incubated with EGF and EGFR-specific mAb. (h,i) Effect of EGF, EGFR-specific mAb and Erlotinib on HCV infection in PHH. Intracellular HCV RNA in PHH infected with (h) HCVcc or (i) serum-derived HCV (one representative experiment) was measured by qRT-PCR. **, P<0.005; ***, P<0.0005. Unless otherwise indicated: EGFR-specific and control mAbs: 10 μg mL−1, EGF: 1 μg mL−1, ERL= Erlotinib: 10 μM.
Fig. 4
Fig. 4. EGFR mediates HCV entry at postbinding steps by promoting CD81-CLDN1 co-receptor interactions and membrane fusion
(a,b) HCV entry factor expression after RTK silencing or PKI treatment. (a) Cell surface expression of entry factors in EGFR or EphA2-silenced Huh7.5.1 cells assessed by flow cytometry. SR-BI silencing served as positive control. (b) Western blot analysis of HCV entry factor expression in PKI- or siRNA-treated Huh7.5.1 cells. (ce) Effect of Erlotinib and EGFR-specific mAb on HCV binding and postbinding steps. (c) Flow cytometric analysis of HCV glycoprotein sE2-binding to Huh7.5.1 cells incubated with EGFR-specific mAb or transfected with siEGFR. SR-BI-specific reagents served as positive controls. (d) HCVcc infection of Huh7.5.1 cells and (e) HCVpp entry into PHH after inhibition of binding and postbinding steps by the indicated compounds (EGFR-specific mAbs: 10 and 50 μg mL−1) (f,g) Effect of Erlotinib and EGF on HCV entry kinetics. Time-course of HCVcc infection of Huh7.5.1 cells following incubation with (f) Erlotinib or indicated compounds or (g) EGF at different time-points during infection (Supplementary Methods). (h) Effect of Erlotinib and EGFR silencing on CD81-CLDN1 association(s). FRET of CD81-CLDN1 co-receptor associations in HepG2-CD81 cells incubated with Erlotinib or EGFR-specific siRNA (means ± SEM). (i) Role of EGFR in viral membrane fusion. Viral glycoprotein-dependent fusion of 293T with Huh7 cells incubated with EGF, Erlotinib or EGFR-specific siRNA was assessed as described25]. *, P<0.05; ***, P<0.0005. Unless otherwise indicated: EGFR-specific and control mAb: 10 μg mL−1, EGF: 1 μg mL−1, Erlotinib: 10 μM.
Fig. 5
Fig. 5. Functional role of EGFR in viral cell-cell transmission and spread
(a) Experimental set-up. HCV producer cells (Pi = HCV RNA-electroporated Huh7.5.1) co-cultivated with non-infected target cells (T = GFP-expressing Huh7.5) [26] were incubated with siEGFR or PKIs. Cell-free HCV transmission was blocked by an E2-neutralizing antibody (25 μg mL−1) [26]. HCV-infected target cells (Ti = GFP+, HCV NS5A+) were quantified by flow cytometry [26]. (b) Immunofluorescence analysis of Pi, T and Ti cells stained with an NS5A-specific antibody. (c) Infectivity of Pi-T cell co-cultivation supernatants (cell-free HCV transmission). (d,e) Quantification of infected Ti cells during Erlotinib (ERL, 10 μM) treatment in the absence (total transmission) and presence (cell-cell transmission) of E2-specific antibody by flow cytometry. (f) Effect of PKIs on viral spread. Long-term HCVcc infection of Huh7.5.1 cells incubated with Erlotinib 48 h post-infection at the indicated concentrations. Medium with solvent (CTRL) or PKI was replenished every 2nd day. Cell viability was assessed using MTT test. (g) EGFR expression in target cells with silenced EGFR expression. Cell surface EGFR expression was analyzed by flow cytometry and target cells were divided in three groups displaying high, medium and low EGFR expression. (h) HCV infection in GFP-positive target cells expressing EGFR at high, medium and low levels (see panel g) assessed as described above. (i) Effect of EGFR silencing on viral spread. Long-term analysis of HCVcc infection in Huh7.5.1 cells transfected with EGFR-specific or control siRNA 24 h post-infection. Cell viability was assessed using MTT test.*, P<0.05; **, P<0.005; ***, P<0.0005.
Fig. 6
Fig. 6. Erlotinib modulates HCV kinetics and inhibits infection in vivo
Chimeric uPA/SCID mice repopulated with PHH [27,28] were treated with Erlotinib or placebo during infection with patient-derived HCV as indicated by the bar and dashed lines. Erlotinib administration and dosage were performed as previously described for xenograft tumor mouse models [30]. Serum HCV load was analyzed at the time points indicated. Results are shown as median viral load of Erlotinib- (n=4) or placebo-treated control mice (n=3). (a,b) Two independent studies (with a total of 2 × 7 mice, respectively) are shown. (c) Pooled data of results shown in a,b (n=14); *, P<0.05.

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