Characterization of the antiviral activity of 4-aminoquinoline derivatives on the Hepatitis C Virus

L’infection par le virus de l’hépatite C (HCV) est un problème majeur de santé publique dont le traitement repose sur l’association d’interféron-α (IFN-α) pégylé et de ribavirine, toutes deux non spécifiques du HCV. Depuis 2011, des inhibiteurs de la protéase virale (boceprevir et telaprevir) ont amélioré l’efficacité du traitement, mais ceci reste limité au virus de génotype 1. De plus, le coût prohibitif et l’importance des leurs effets secondaires oblige au développement de nouveaux composés.La chloroquine, un antipaludique, est capable d’inhiber le HCV, mais son utilisation se complique par divers effets secondaires. Des dérivés ont donc été développés dont certains ont testé au cours de cette thèse. Ainsi un premier composé, la ferroquine (FQ), inhibe l’infection par le HCV à l’étape de fusion et à celle de réplication, mais à de plus fortes concentrations. L’effet sur l’entrée virale a été conforté par la sélection d’un mutant de résistance révélant qu’une simple mutation (S327A) suffit à conférer une résistance à la FQ. L’utilisation de pseudoparticules contenant les glycoprotéines d’enveloppe du HCV a montré que cette inhibition est indépendante du génotype viral. Un autre dérivé testé, compatible avec un potentiel développement thérapeutique, inhibe également l’entrée et la réplication virale. De plus, ce composé, tout comme la FQ, inhibe la transmission cellule-cellule. Enfin, en combinaison avec le boceprevir ou l’IFN-α il présente un effet antiviral additif. Ce dernier résultat a également été retrouvé pour la FQ.Hepatitis C virus (HCV) is a major cause of chronic liver disease for which standard-of-care treatment consisted in a bi-therapy based on two non-specific inhibitors : pegylated interferon-α (IFN-α) and ribavirin. Since 2011 protease inhibitors (boceprevir and telaprevir) increased the response to treatment. However, they are specific of genotype 1. Furthermore, they cause severe side effects and their cost remains high, leading to the development of new compounds.Chloroquine, usually used against malaria, is able to inhibit HCV. But its clinical use is complicated by several side effects. That is why chloroquine derivatives have been developed and tested during this thesis. A first compound, the ferroquine (FQ) inhibit HCV infection at fusion step and also replication, albeit at higher concentrations. The effect of FQ on HCV entry was confirmed by the selection of a resistant mutant showing that a single mutation (S327A) can confer resistance to FQ. The use of pseudoparticles harboring E1E2 glycoproteins at their surface showed this inhibition is not genotype specific. An other derivative,which pharmacodynamics properties would allow some potential therapeutic use, has been tested, showing an inhibition of entry and replication steps. As FQ, this compound is able to inhibit cell-to-cell transmission of HCV and has, in combination with boceprevir or IFN-α, additive antiviral effects against HCV

Caractérisation de l'activité antivirale de dérivés de 4-aminoquinolines sur le virus de l'hépatite C

L infection par le virus de l hépatite C (HCV) est un problème majeur de santé publique dont le traitement repose sur l association d interféron-a (IFN-a) pégylé et de ribavirine, toutes deux non spécifiques du HCV. Depuis 2011, des inhibiteurs de la protéase virale (boceprevir et telaprevir) ont amélioré l efficacité du traitement, mais ceci reste limité au virus de génotype 1. De plus, le coût prohibitif et l importance des leurs effets secondaires oblige au développement de nouveaux composés.La chloroquine, un antipaludique, est capable d inhiber le HCV, mais son utilisation se complique par divers effets secondaires. Des dérivés ont donc été développés dont certains ont testé au cours de cette thèse. Ainsi un premier composé, la ferroquine (FQ), inhibe l infection par le HCV à l étape de fusion et à celle de réplication, mais à de plus fortes concentrations. L effet sur l entrée virale a été conforté par la sélection d un mutant de résistance révélant qu une simple mutation (S327A) suffit à conférer une résistance à la FQ. L utilisation de pseudoparticules contenant les glycoprotéines d enveloppe du HCV a montré que cette inhibition est indépendante du génotype viral. Un autre dérivé testé, compatible avec un potentiel développement thérapeutique, inhibe également l entrée et la réplication virale. De plus, ce composé, tout comme la FQ, inhibe la transmission cellule-cellule. Enfin, en combinaison avec le boceprevir ou l IFN-a il présente un effet antiviral additif. Ce dernier résultat a également été retrouvé pour la FQ.Hepatitis C virus (HCV) is a major cause of chronic liver disease for which standard-of-care treatment consisted in a bi-therapy based on two non-specific inhibitors : pegylated interferon-a (IFN-a) and ribavirin. Since 2011 protease inhibitors (boceprevir and telaprevir) increased the response to treatment. However, they are specific of genotype 1. Furthermore, they cause severe side effects and their cost remains high, leading to the development of new compounds.Chloroquine, usually used against malaria, is able to inhibit HCV. But its clinical use is complicated by several side effects. That is why chloroquine derivatives have been developed and tested during this thesis. A first compound, the ferroquine (FQ) inhibit HCV infection at fusion step and also replication, albeit at higher concentrations. The effect of FQ on HCV entry was confirmed by the selection of a resistant mutant showing that a single mutation (S327A) can confer resistance to FQ. The use of pseudoparticles harboring E1E2 glycoproteins at their surface showed this inhibition is not genotype specific. An other derivative,which pharmacodynamics properties would allow some potential therapeutic use, has been tested, showing an inhibition of entry and replication steps. As FQ, this compound is able to inhibit cell-to-cell transmission of HCV and has, in combination with boceprevir or IFN-a, additive antiviral effects against HCV.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro

International audienceThe clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated

The antimalarial ferroquine is an inhibitor of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. Conclusion: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals

Author Correction: SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses.

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV

CLDN6/R209Q and OCLN/P24A mutations do not affect the kinetics of HCV entry and cellular physiology.

<p>Huh-7 cells were transduced with CLDN6/wt or CLDN6/R209Q and OCLN/wt or OCLN/P24A either alone or in combination. (<b>A</b>) Forty-eight hours after the last transduction round, cells were infected with HCVcc for 2h, 1h30, 1h or 30min. After infection, cells were rinsed and fresh medium was added. At 30h post-infection, cells were lysed and <i>Gaussia</i>-luciferase activities were measured. Results were normalized to luciferase activities measured in cells expressing the wt forms of CLDN6 and OCLN. (<b>B</b>) Cells were infected with HCVcc at indicated m.o.i. in serum-free DMEM for 2 h. At 30 h post-infection, cells were lysed and <i>Gaussia</i>-luciferase activities were measured. Results were normalized to luciferase activities measured in cells expressing the wt forms of CLDN6 and OCLN. Results are presented as mean ± SD of two independent experiments. (<b>C</b>) Huh-7-Lunet-CD81-FLuc cells, which endogenously expressed the <i>Firefly</i>-luciferase reporter gene, were transduced with CLDN6/wt or CLDN6/R209Q and OCLN/wt or OCLN/P24A either alone or in combination. Forty-eight hours after the last transduction round, cells were infected with HCVcc expressing the <i>Gaussia</i>-luciferase reporter gene. At 30h post-infection, cells were lysed and luciferase activities were measured with the Dual-luciferase^® reporter assay system (Promega). <i>Gaussia</i>-luciferase activities were normalized to Huh-7-Lunet-CD81 cells <i>Firefly</i>-luciferase activities. Results were adjusted to 100% infection for cells expressing the wt forms of CLDN6 and OCLN. Results are presented as mean ± SD of at least three independent experiments.</p

OCLN/P24A mutation does not affect HCVpp entry.

<p>786-O cells were transduced twice at 24h interval with OCLN/wt or OCLN/P24A. Expression was analysed 48h after the last transduction round. (A) OCLN/wt and OCLN/P24A expression in 786-O cells was estimated through the percentage of GFP positive cells by flow cytometry analysis. (B) OCLN/wt and OCLN/P24A export at the cell surface in transduced 786-O cells was analysed by microscopy. (<b>C</b>) Two days after the last transduction, 786-O cells were infected with HCVpp1a, HCVpp2a or VSVpp expressing the <i>Firefly</i>-luciferase reporter gene. Non-transduced 786-O cells (Mock) were infected in parallel. Results are presented in relative luciferase units (RLU). (<b>D</b>) 786-O cells were transduced sequentially with a combination of lentivectors encoding SRB1, CD81 and CLDN1 in addition to OCLN/wt or OCLN/P24A. Cells were infected 48h after the last transduction round with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing OCLN/wt. (<b>E</b>) 786-O cells transduced to express SRB1, CD81 and CLDN1 in addition to OCLN/wt or OCLN/P24A were infected with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing OCLN/wt. Results are presented as mean ± SD of four independent experiments. *** means a <i>p</i> value below 0.001.</p

Identification of a new benzimidazole derivative as an antiviral against hepatitis C virus

Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle

CLDN6/R209Q mutation does not affect HCVpp entry.

<p>293T cells were transduced twice at 24h interval with CLDN6/wt or CLDN6/R209Q. Expression was analysed 48h after the last transduction round. (A) CLDN6/wt and CLDN6/R209Q cell surface expression was measured by flow cytometry using an anti-CLDN6 antibody, and compared to that of non-transduced 293T cells (293T Mock). Cells stained with secondary antibodies were used as negative controls (dashed line). (B) CLDN6/wt and CLDN6/R209Q expression in 293T cells was also analyzed by immunofluorescence with an anti-CLDN6 antibody. (<b>C</b>) Two days after the last transduction, 293T cells were infected with HCVpp1a, HCVpp2a or VSVpp expressing the <i>Firefly</i>-luciferase reporter gene. Non-transduced 293T cells (Mock) were infected in parallel. Results are presented in relative luciferase units (RLU). (D) 293T cells were transduced sequentially with a combination of lentivectors encoding SRB1, CD81 and/or OCLN in addition to CLDN6/wt or CLDN6/R209Q. Cells were next infected with HCVpp1a or VSVpp 48h after the last transduction round. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing CLDN6/wt. (<b>E</b>) 293T cells transduced to express SRB1, CD81 and OCLN in addition to CLDN6/wt or CLDN6/R209Q were infected with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing CLDN6/wt. Results are presented as mean ± SD of three independent experiments. *** means a <i>p</i> value below 0.001.</p