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Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

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Netrin-1 increases HCV through the UNC5A receptor.Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05), while siRNA-mediated knockdown was quantified for each UNC5 transcript on days four and eight post-infection by RT-qPCR (right-hand graphs; data shown as mean ± standard deviation, n = 3). The underlying data for panels in this figure can be found in S1 Data.
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pbio.1002421.g011: Netrin-1 increases HCV through the UNC5A receptor.Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05), while siRNA-mediated knockdown was quantified for each UNC5 transcript on days four and eight post-infection by RT-qPCR (right-hand graphs; data shown as mean ± standard deviation, n = 3). The underlying data for panels in this figure can be found in S1 Data.

Mentions: Based on these results, we set out to identify which of the UNC5 receptors was responsible for mediating the effects of Netrin-1 by monitoring HCV in Netrin-1-Fc-treated Huh7.5 cells, which had previously been subjected to siRNA-mediated depletion of each individual UNC5 transcript. Depletion of UNC5A alone induced an up to 4-fold increase in the levels of HCV (Fig 11, left column). RT-qPCR conducted to detect the UNC5 transcripts confirmed the efficacy of the siRNAs (Fig 11, right column). These results were subsequently validated using RNAi-based depletion and plasmid-mediated forced expression approaches of UNC5A (Uniprot Acc. # Q6ZN44) on viral parameters. Indeed, intra/extracellular infectivity parameters increased and decreased by 3-fold to 6-fold upon UNC5A depletion or overexpression, respectively (S14 Fig). These results demonstrate that Netrin-1 exerts its pro-HCV effect via inhibition of the UNC5A receptor that itself decreases Netrin-1’s proviral effect. They also indicate that UNC5A-related functions ultimately condition infectivity of the virus particle, through increased viral propagation inducing enhanced Netrin-1 expression.


Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

Netrin-1 increases HCV through the UNC5A receptor.Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05), while siRNA-mediated knockdown was quantified for each UNC5 transcript on days four and eight post-infection by RT-qPCR (right-hand graphs; data shown as mean ± standard deviation, n = 3). The underlying data for panels in this figure can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816328&req=5

pbio.1002421.g011: Netrin-1 increases HCV through the UNC5A receptor.Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05), while siRNA-mediated knockdown was quantified for each UNC5 transcript on days four and eight post-infection by RT-qPCR (right-hand graphs; data shown as mean ± standard deviation, n = 3). The underlying data for panels in this figure can be found in S1 Data.
Mentions: Based on these results, we set out to identify which of the UNC5 receptors was responsible for mediating the effects of Netrin-1 by monitoring HCV in Netrin-1-Fc-treated Huh7.5 cells, which had previously been subjected to siRNA-mediated depletion of each individual UNC5 transcript. Depletion of UNC5A alone induced an up to 4-fold increase in the levels of HCV (Fig 11, left column). RT-qPCR conducted to detect the UNC5 transcripts confirmed the efficacy of the siRNAs (Fig 11, right column). These results were subsequently validated using RNAi-based depletion and plasmid-mediated forced expression approaches of UNC5A (Uniprot Acc. # Q6ZN44) on viral parameters. Indeed, intra/extracellular infectivity parameters increased and decreased by 3-fold to 6-fold upon UNC5A depletion or overexpression, respectively (S14 Fig). These results demonstrate that Netrin-1 exerts its pro-HCV effect via inhibition of the UNC5A receptor that itself decreases Netrin-1’s proviral effect. They also indicate that UNC5A-related functions ultimately condition infectivity of the virus particle, through increased viral propagation inducing enhanced Netrin-1 expression.

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

Show MeSH
Related in: MedlinePlus