Functional characterization of virus-kinase interactions during cellular entry of hepatitis C virus

Le virus de l'hépatite C (HCV) est une cause majeure de maladie chronique du foie et de carcinome hépatocellulaire. Les options thérapeutiques pour traiter l'hépatite chronique sont limitées par des coûts élevés, des effets secondaires et une résistance virale. L'entrée du HCV est la première étape d'interaction entre le virus et la cellule hôte. Elle est requise pour l'initiation, la propagation et le maintien de l'infection, ce qui en fait une cible prometteuse pour les traitements antiviraux. L’entrée du HCV nécessite l'interaction coopérative de plusieurs facteurs cellulaires, y compris CD81 et claudine-1 (CLDN1). Nous avons récemment identifié un rôle pour le récepteur à l’EGF (EGFR) et le récepteur à l’ephrine A2 (EphA2) dans l'entrée du HCV par la régulation de la formation du complexe de co-récepteurs CD81-CLDN1, ce qui suggère que la signalisation de ces récepteurs joue un rôle dans l'entrée du virus. Nous avons voulu identifier les mécanismes moléculaires de signalisation de l’EGFR requis pour l'entrée du HCV et avons identifié HRas comme un transducteur de signalisation clé de l'hôte. Des études d'imagerie ont révélées que la signalisation de HRas peut moduler la diffusion et le trafic membranaire de CD81, ce qui permet l’assemblage du complexe de récepteurs. De plus, HRas s’associe avec les récepteurs de l'hôte CD81 et CLDN1 et des facteurs d’entrée du HCV inconnus jusque là: l’intégrine beta1 et Rap2B. Le HCV profite donc de la signalisation de HRas pour l'entrée cellulaire. Ces données améliorent notre compréhension des mécanismes moléculaires de l'entrée du HCV induite par l’EGFR et ouvrent de nouvelles perspectives pour le développement d'antiviraux.Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Therapeutic options to treat chronic viral hepatitis are limited by high costs, side effects and viral resistance in most patients. HCV entry is the first step of interaction between the virus and the host cell. It is required for the initiation, propagation and maintenance of infection, making it a promising target for antiviral therapy. HCV entry requires the cooperative interaction of several cellular factors, including CD81 and claudin-1 (CLDN1). We have recently identified a role for EGF receptor (EGFR) and ephrin receptor A2 (EphA2) in HCV entry by regulating the formation of the co-receptor complex CD81-CLDN1, suggesting that the signaling of these receptors might play a role in viral entry. However, the precise mechanisms of regulation are unknown. We wanted to identify the molecular mechanisms of EGFR signaling required for the HCV entry process. We identify HRas as key host signaling transducer for HCV entry. Advanced imaging studies have revealed that HRas signaling can modulate the lateral diffusion and membrane trafficking of CD81. A modified diffusion of CD81 allows the assembly of the receptors complex. In addition, HRas associates with tetraspanin microdomains containing the host receptors CD81 and CLDN1 and HCV entry factors previously unknown: the integrin beta1 and Rap2B. HCV therefore exploits HRas signaling for cellular entry. These data improve our understanding of the molecular mechanisms of HCV entry induced by EGFR and open new perspectives for the development of antivirals targeting signaling pathways

Caractérisation fonctionnelle des interactions virus-kinases lors de l'entrée cellulaire du virus de l'hépatite C

Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Therapeutic options to treat chronic viral hepatitis are limited by high costs, side effects and viral resistance in most patients. HCV entry is the first step of interaction between the virus and the host cell. It is required for the initiation, propagation and maintenance of infection, making it a promising target for antiviral therapy. HCV entry requires the cooperative interaction of several cellular factors, including CD81 and claudin-1 (CLDN1). We have recently identified a role for EGF receptor (EGFR) and ephrin receptor A2 (EphA2) in HCV entry by regulating the formation of the co-receptor complex CD81-CLDN1, suggesting that the signaling of these receptors might play a role in viral entry. However, the precise mechanisms of regulation are unknown. We wanted to identify the molecular mechanisms of EGFR signaling required for the HCV entry process. We identify HRas as key host signaling transducer for HCV entry. Advanced imaging studies have revealed that HRas signaling can modulate the lateral diffusion and membrane trafficking of CD81. A modified diffusion of CD81 allows the assembly of the receptors complex. In addition, HRas associates with tetraspanin microdomains containing the host receptors CD81 and CLDN1 and HCV entry factors previously unknown: the integrin beta1 and Rap2B. HCV therefore exploits HRas signaling for cellular entry. These data improve our understanding of the molecular mechanisms of HCV entry induced by EGFR and open new perspectives for the development of antivirals targeting signaling pathways.Le virus de l'hépatite C (HCV) est une cause majeure de maladie chronique du foie et de carcinome hépatocellulaire. Les options thérapeutiques pour traiter l'hépatite chronique sont limitées par des coûts élevés, des effets secondaires et une résistance virale. L'entrée du HCV est la première étape d'interaction entre le virus et la cellule hôte. Elle est requise pour l'initiation, la propagation et le maintien de l'infection, ce qui en fait une cible prometteuse pour les traitements antiviraux. L’entrée du HCV nécessite l'interaction coopérative de plusieurs facteurs cellulaires, y compris CD81 et claudine-1 (CLDN1). Nous avons récemment identifié un rôle pour le récepteur à l’EGF (EGFR) et le récepteur à l’ephrine A2 (EphA2) dans l'entrée du HCV par la régulation de la formation du complexe de co-récepteurs CD81-CLDN1, ce qui suggère que la signalisation de ces récepteurs joue un rôle dans l'entrée du virus. Nous avons voulu identifier les mécanismes moléculaires de signalisation de l’EGFR requis pour l'entrée du HCV et avons identifié HRas comme un transducteur de signalisation clé de l'hôte. Des études d'imagerie ont révélées que la signalisation de HRas peut moduler la diffusion et le trafic membranaire de CD81, ce qui permet l’assemblage du complexe de récepteurs. De plus, HRas s’associe avec les récepteurs de l'hôte CD81 et CLDN1 et des facteurs d’entrée du HCV inconnus jusque là: l’intégrine beta1 et Rap2B. Le HCV profite donc de la signalisation de HRas pour l'entrée cellulaire. Ces données améliorent notre compréhension des mécanismes moléculaires de l'entrée du HCV induite par l’EGFR et ouvrent de nouvelles perspectives pour le développement d'antiviraux

Caractérisation fonctionnelle des interactions virus-kinases lors de l'entrée cellulaire du virus de l'hépatite C

Le virus de l'hépatite C (HCV) est une cause majeure de maladie chronique du foie et de carcinome hépatocellulaire. Les options thérapeutiques pour traiter l'hépatite chronique sont limitées par des coûts élevés, des effets secondaires et une résistance virale. L'entrée du HCV est la première étape d'interaction entre le virus et la cellule hôte. Elle est requise pour l'initiation, la propagation et le maintien de l'infection, ce qui en fait une cible prometteuse pour les traitements antiviraux. L entrée du HCV nécessite l'interaction coopérative de plusieurs facteurs cellulaires, y compris CD81 et claudine-1 (CLDN1). Nous avons récemment identifié un rôle pour le récepteur à l EGF (EGFR) et le récepteur à l ephrine A2 (EphA2) dans l'entrée du HCV par la régulation de la formation du complexe de co-récepteurs CD81-CLDN1, ce qui suggère que la signalisation de ces récepteurs joue un rôle dans l'entrée du virus. Nous avons voulu identifier les mécanismes moléculaires de signalisation de l EGFR requis pour l'entrée du HCV et avons identifié HRas comme un transducteur de signalisation clé de l'hôte. Des études d'imagerie ont révélées que la signalisation de HRas peut moduler la diffusion et le trafic membranaire de CD81, ce qui permet l assemblage du complexe de récepteurs. De plus, HRas s associe avec les récepteurs de l'hôte CD81 et CLDN1 et des facteurs d entrée du HCV inconnus jusque là: l intégrine beta1 et Rap2B. Le HCV profite donc de la signalisation de HRas pour l'entrée cellulaire. Ces données améliorent notre compréhension des mécanismes moléculaires de l'entrée du HCV induite par l EGFR et ouvrent de nouvelles perspectives pour le développement d'antiviraux.Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Therapeutic options to treat chronic viral hepatitis are limited by high costs, side effects and viral resistance in most patients. HCV entry is the first step of interaction between the virus and the host cell. It is required for the initiation, propagation and maintenance of infection, making it a promising target for antiviral therapy. HCV entry requires the cooperative interaction of several cellular factors, including CD81 and claudin-1 (CLDN1). We have recently identified a role for EGF receptor (EGFR) and ephrin receptor A2 (EphA2) in HCV entry by regulating the formation of the co-receptor complex CD81-CLDN1, suggesting that the signaling of these receptors might play a role in viral entry. However, the precise mechanisms of regulation are unknown. We wanted to identify the molecular mechanisms of EGFR signaling required for the HCV entry process. We identify HRas as key host signaling transducer for HCV entry. Advanced imaging studies have revealed that HRas signaling can modulate the lateral diffusion and membrane trafficking of CD81. A modified diffusion of CD81 allows the assembly of the receptors complex. In addition, HRas associates with tetraspanin microdomains containing the host receptors CD81 and CLDN1 and HCV entry factors previously unknown: the integrin beta1 and Rap2B. HCV therefore exploits HRas signaling for cellular entry. These data improve our understanding of the molecular mechanisms of HCV entry induced by EGFR and open new perspectives for the development of antivirals targeting signaling pathways.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

A Novel Monoclonal Anti-CD81 Antibody Produced by Genetic Immunization Efficiently Inhibits Hepatitis C Virus Cell-Cell Transmission

Background and Aims: Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. Methods: Using genetic immunization, we produced four monoclonal antibodies (mAbs) against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines. Results: The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity. Conclusion: A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HC

Functional analysis of claudin-6 and claudin-9 as entry factors for hepatitis C virus infection of human hepatocytes using monoclonal antibodies.

International audience: The relevance of claudin-6 and claudin-9 in hepatitis C virus (HCV) entry remains elusive. We produced claudin-6 or claudin-9 specific monoclonal antibodies that inhibit HCV entry into non-hepatic cells expressing exogenous claudin-6 or claudin-9. These antibodies had no effect on HCV infection of hepatoma cells or primary hepatocytes. Thus although claudin-6 and claudin-9 can serve as entry factors in cell lines, HCV infection into human hepatocytes is not dependent on claudin-6 and claudin-9

Synergistic effect of anti-envelope and anti-CD81 antibodies on inhibition of HCV infection.

<p>HCVpp of strains P02VJ or HCVcc-Luc Jc1 were pre-incubated with anti-E2 mAb IGH461 or purified heterologous anti-HCV IgG (1 or 10 µg/ml) obtained from an unrelated chronically infected subject or isotype control IgG for 1 hour at 37°C and added to Huh7.5.1 cells pre-incubated with serial dilutions of anti-CD81 QV-6A8-F2-C4 or rat isotype control mAbs. HCVpp entry and HCVcc infection were analyzed by luciferase assay. The Combination Index (CI) was calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#pone.0064221-Zhu1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#pone.0064221-Zhao1" target="_blank">[43]</a>. A CI less than 0.9, between 0.9 and 1.1, and more than 1.1 indicates synergy, additivity, and antagonism, respectively. CI for anti-CD81 mAb in combination with 10 µg/ml anti-HCV IgG in HCVpp entry inhibition was calculated for an IC₇₅ as the combination resulted in an inhibition below the IC₅₀ and is indicated by a star (*). IC₅₀ of anti-envelope antibodies: anti-E2, 70±5 µg/ml (for HCVpp); anti-HCV IgG, 40±3 µg/ml (for HCVpp), 120±6 µg/ml (for HCVcc).</p

Anti-CD81 mAb inhibits HCV cell-to-cell transmission and viral spread.

<p>(A) Quantification of HCV-infected target cells (Ti) after co–cultivation with HCV producer cells (Pi) during incubation with control or anti-CD81 QV-6A8-F2-C4 mAbs (10 µg/ml) in the presence of neutralizing anti-HCV E2 mAb (AP33, 25 µg/ml) by flow cytometry. (B) Percentage of infected target cells is shown as histograms and is represented as means ± SD from three experiments. (C) Long-term analysis of HCVcc infection in the presence or absence of control or anti-CD81 QV-6A8-F2-C4 mAbs at the indicated concentrations. Antibodies were added 48 h after HCVcc infection and control medium or medium containing mAbs were replenished every 4 days. Luciferase activity was determined in cell lysates every 2 days. Data are expressed as Log₁₀ RLU and represent means ± SD of three experiments performed in duplicate. (D) Cell viability after long-term exposure to anti-CD81 mAb QV-6A8-F2-C4. Cell viability was assessed using MTT assay after incubation of Huh7.5.1 cells for 14 days in the presence or absence of control or anti-CD81 mAbs at 1, 10, or 100 µg/ml. Data are expressed as % cell viability relative to cells incubated in the absence of mAb and represent means ± SD from one experiment performed in triplicate. (E–F) Virus spread in the presence or absence of anti-CD81 mAbs QV-6A8-F2-C4 (E) and JS81 (F). Antibodies (50 µg/ml) were added 48 h after HCVcc (Jc1) infection and control medium or medium containing antibodies were replenished every 4 days. HCV-infected cells were visualized 9 days post-infection via immunofluorescence using anti-NS5A (E) or anti-E2 (CBH23) (F) antibodies. The percentage of infected cells was calculated as the number of infected cells relative to the total number of cells as assessed by 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei.</p

Anti-CD81 mAbs dose-dependently inhibit HCV infection.

<p>(A–B) Dose-dependent inhibition of HCV infection by anti-CD81 mAbs. Huh7.5.1 cells were pre-incubated with increasing concentrations of anti-CD81 or isotype control (CTRL IgG) mAbs for 1 h at 37°C before infection with (A) HCVcc (Luc-Jc1 (2a)) or (B) HCVpp (HCV-J (1b)). Three days later, viral infection was quantitated by assessing the expression of luciferase reporter gene. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#s3" target="_blank">Results</a> are expressed as % HCVcc infection or % HCVpp entry and represent means ± SD of one representative experiment performed in triplicate. (C) Inhibition of infection of HCVpp bearing envelope glycoproteins from genotypes 1–6. Huh7.5.1 cells were pre-incubated with a fixed concentration (100 µg/ml) of antibodies before infection with HCVpp (strains H77 (1a), JFH1 (2a), UKN3A1.28 (3a), UKN4.21.16 (4), UKN5.14.4 (5), UKN6.5.340 (6)). Means ± SD from a representative experiment performed in triplicate are shown.</p

Synergy between anti-CD81 and anti-envelope antibodies in inhibiting HCV infection.

<p>(A–B) Patient derived HCVpp P02VJ or (C) HCVcc-Luc Jc1 were pre-incubated with (A) anti-E2 mAb (IGH461) or (B–C) purified heterologous anti-HCV IgG (1 or 10 µg/ml) obtained from an unrelated chronically infected subject or isotype control IgG for 1 h at 37°C and added to Huh7.5.1 cells that had been pre-incubated with increasing concentrations of control or anti-CD81 mAb QV-6A8-F2-C4. In anti-envelope antibody monotherapy setting, HCVpp or HCVcc were in parallel pre-incubated with increasing concentrations of anti-E2 mAb or anti-HCV IgG. HCVpp entry and HCVcc infection were analyzed by luciferase assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#s3" target="_blank">Results</a> are expressed as % HCVpp entry or HCVcc infection and represent means ± SD from a representative experiment performed in triplicate.</p

CD81-Receptor Associations — Impact for Hepatitis C Virus Entry and Antiviral Therapies

Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection