Limits...
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.

Show MeSH

Related in: MedlinePlus

Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro.A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B. Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). C. Supernatant infectivity was quantified on days two and four post-infection by the TCID50 method (n = 3, Mann-Whitney test, p < 0.05.). D. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). E. Density of infectivity values was quantified for each collected sucrose gradient fraction, 3 d post-infection, by the TCID50 method (n = 3). Buoyant densities were determined by refractometry. F. Netrin-1 increases the specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio and refractometry (n = 3). G. Assessment of Netrin-1 mRNA knockdown. Cells were initially transfected with control and Netrin-1-specific siRNAs, then harvested at the indicated time points and processed for Netrin-1 mRNA quantification by RT-qPCR or by immunoblotting in microsomes using an anti-Netrin-1 antibody (data are shown as mean ± standard deviation, n = 3, Mann-Whitney test, p < 0.05). H. Netrin-1 depletion impedes HCV. Huh7.5 cells were transfected with siRNAs, infected at a MOI of 0.1 24 h after seeding, and trypsinized at day five post-infection before a second siRNA transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). I. Supernatant infectivity was quantified on days one, four, seven, and nine post-infection by the TCID50 method (n = 3, Wilcoxon test, p < 0.05). J. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). K. 6 d post-infection, culture supernatants were subjected to sucrose gradient centrifugation, and infectivity was quantified in each collected fraction by the TCID50 method. Buoyant densities were determined by refractometry (n = 3). L. Netrin-1 depletion decreases specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio. (n = 3). The underlying data for panels in this figure can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4816328&req=5

pbio.1002421.g009: Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro.A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B. Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). C. Supernatant infectivity was quantified on days two and four post-infection by the TCID50 method (n = 3, Mann-Whitney test, p < 0.05.). D. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). E. Density of infectivity values was quantified for each collected sucrose gradient fraction, 3 d post-infection, by the TCID50 method (n = 3). Buoyant densities were determined by refractometry. F. Netrin-1 increases the specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio and refractometry (n = 3). G. Assessment of Netrin-1 mRNA knockdown. Cells were initially transfected with control and Netrin-1-specific siRNAs, then harvested at the indicated time points and processed for Netrin-1 mRNA quantification by RT-qPCR or by immunoblotting in microsomes using an anti-Netrin-1 antibody (data are shown as mean ± standard deviation, n = 3, Mann-Whitney test, p < 0.05). H. Netrin-1 depletion impedes HCV. Huh7.5 cells were transfected with siRNAs, infected at a MOI of 0.1 24 h after seeding, and trypsinized at day five post-infection before a second siRNA transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). I. Supernatant infectivity was quantified on days one, four, seven, and nine post-infection by the TCID50 method (n = 3, Wilcoxon test, p < 0.05). J. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). K. 6 d post-infection, culture supernatants were subjected to sucrose gradient centrifugation, and infectivity was quantified in each collected fraction by the TCID50 method. Buoyant densities were determined by refractometry (n = 3). L. Netrin-1 depletion decreases specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio. (n = 3). The underlying data for panels in this figure can be found in S1 Data.

Mentions: These initial findings prompted us to investigate whether Netrin-1 produced by cultured cells was, in turn, able to promote HCV replication and/or propagation. HCV-infected proliferative Huh7.5 cells were transfected with a Netrin-1 expression plasmid. Intracellular HCV RNA, viral RNA release, supernatant infectivity, and virion-specific infectivity were then monitored. The expression vector produced a modified Netrin-1 containing the hemagglutinin (HA) epitope at its carboxyl-terminus, while a plasmid expressing HA-tagged vanilloid receptor (VR1-HA) served as a control. Immunoblotting analyses confirmed expression of both proteins in the transfected cells (Fig 9A). Plasmid-delivered Netrin-1-HA produced a 3-fold increase in the intracellular HCV RNA compared to cells transfected with VR1-HA (Fig 9B) and was accompanied by a 2.5-fold increase in supernatant infectivity, measured using the TCID50 protocol (Fig 9C). Furthermore, overexpression of Netrin-1 resulted in a 2-fold increase in the level of intracellular infectivity (Fig 9D) and likewise increased the infectivity peak of the released virions (Fig 9E). Since LARP1 intriguingly concentrates at the ER and in the proximity of lipid droplets, an important viral budding site [38], upon infection, we tested whether accumulation of Netrin-1 in microsomes could foster increased morphogenesis, through the evaluation of the specific infectivity of virions. This viral parameter is defined by the ratio of biological infectivity values, expressed as TCID50 units, and viral RNA copy numbers of the sample. To achieve this, we plotted TCID50/extracellular HCV RNA ratios against their buoyant densities for Netrin-1- and control VR1-transfected samples. Netrin-1 overexpression caused a 4-fold increase in the specific infectivity of released virions (Fig 9F), suggesting that the protein induces alterations in the virus particle. Netrin-1 also resulted in a viral increase of the poorly infectious, high-density fractions (S4 Fig).


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 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro.A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B. Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). C. Supernatant infectivity was quantified on days two and four post-infection by the TCID50 method (n = 3, Mann-Whitney test, p < 0.05.). D. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). E. Density of infectivity values was quantified for each collected sucrose gradient fraction, 3 d post-infection, by the TCID50 method (n = 3). Buoyant densities were determined by refractometry. F. Netrin-1 increases the specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio and refractometry (n = 3). G. Assessment of Netrin-1 mRNA knockdown. Cells were initially transfected with control and Netrin-1-specific siRNAs, then harvested at the indicated time points and processed for Netrin-1 mRNA quantification by RT-qPCR or by immunoblotting in microsomes using an anti-Netrin-1 antibody (data are shown as mean ± standard deviation, n = 3, Mann-Whitney test, p < 0.05). H. Netrin-1 depletion impedes HCV. Huh7.5 cells were transfected with siRNAs, infected at a MOI of 0.1 24 h after seeding, and trypsinized at day five post-infection before a second siRNA transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). I. Supernatant infectivity was quantified on days one, four, seven, and nine post-infection by the TCID50 method (n = 3, Wilcoxon test, p < 0.05). J. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). K. 6 d post-infection, culture supernatants were subjected to sucrose gradient centrifugation, and infectivity was quantified in each collected fraction by the TCID50 method. Buoyant densities were determined by refractometry (n = 3). L. Netrin-1 depletion decreases specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio. (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.g009: Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro.A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B. Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). C. Supernatant infectivity was quantified on days two and four post-infection by the TCID50 method (n = 3, Mann-Whitney test, p < 0.05.). D. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). E. Density of infectivity values was quantified for each collected sucrose gradient fraction, 3 d post-infection, by the TCID50 method (n = 3). Buoyant densities were determined by refractometry. F. Netrin-1 increases the specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio and refractometry (n = 3). G. Assessment of Netrin-1 mRNA knockdown. Cells were initially transfected with control and Netrin-1-specific siRNAs, then harvested at the indicated time points and processed for Netrin-1 mRNA quantification by RT-qPCR or by immunoblotting in microsomes using an anti-Netrin-1 antibody (data are shown as mean ± standard deviation, n = 3, Mann-Whitney test, p < 0.05). H. Netrin-1 depletion impedes HCV. Huh7.5 cells were transfected with siRNAs, infected at a MOI of 0.1 24 h after seeding, and trypsinized at day five post-infection before a second siRNA transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data shown as mean ± standard deviation, n = 3, Wilcoxon test, p < 0.05). I. Supernatant infectivity was quantified on days one, four, seven, and nine post-infection by the TCID50 method (n = 3, Wilcoxon test, p < 0.05). J. Intracellular infectivity was quantified on day four post-infection by the TCID50 method (n = 3). K. 6 d post-infection, culture supernatants were subjected to sucrose gradient centrifugation, and infectivity was quantified in each collected fraction by the TCID50 method. Buoyant densities were determined by refractometry (n = 3). L. Netrin-1 depletion decreases specific infectivity of virions. Specific infectivity was calculated for each collected fraction using the TCID50/HCV RNA ratio. (n = 3). The underlying data for panels in this figure can be found in S1 Data.
Mentions: These initial findings prompted us to investigate whether Netrin-1 produced by cultured cells was, in turn, able to promote HCV replication and/or propagation. HCV-infected proliferative Huh7.5 cells were transfected with a Netrin-1 expression plasmid. Intracellular HCV RNA, viral RNA release, supernatant infectivity, and virion-specific infectivity were then monitored. The expression vector produced a modified Netrin-1 containing the hemagglutinin (HA) epitope at its carboxyl-terminus, while a plasmid expressing HA-tagged vanilloid receptor (VR1-HA) served as a control. Immunoblotting analyses confirmed expression of both proteins in the transfected cells (Fig 9A). Plasmid-delivered Netrin-1-HA produced a 3-fold increase in the intracellular HCV RNA compared to cells transfected with VR1-HA (Fig 9B) and was accompanied by a 2.5-fold increase in supernatant infectivity, measured using the TCID50 protocol (Fig 9C). Furthermore, overexpression of Netrin-1 resulted in a 2-fold increase in the level of intracellular infectivity (Fig 9D) and likewise increased the infectivity peak of the released virions (Fig 9E). Since LARP1 intriguingly concentrates at the ER and in the proximity of lipid droplets, an important viral budding site [38], upon infection, we tested whether accumulation of Netrin-1 in microsomes could foster increased morphogenesis, through the evaluation of the specific infectivity of virions. This viral parameter is defined by the ratio of biological infectivity values, expressed as TCID50 units, and viral RNA copy numbers of the sample. To achieve this, we plotted TCID50/extracellular HCV RNA ratios against their buoyant densities for Netrin-1- and control VR1-transfected samples. Netrin-1 overexpression caused a 4-fold increase in the specific infectivity of released virions (Fig 9F), suggesting that the protein induces alterations in the virus particle. Netrin-1 also resulted in a viral increase of the poorly infectious, high-density fractions (S4 Fig).

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