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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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Related in: MedlinePlus

LARP1 relocalizes to the ER upon HCV infection.Cells were infected by HCV for 4 d prior to fixation, LARP1, NS5A, and calnexin staining, and confocal microscopy. A. Li diagrams and Li coefficient (B) calculations for LARP1 and Calnexin expression. Pixels present on the left and right sides of the y-axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the ImageJ software (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given single experiment (n = 3). C. Representative immunofluorescence-based localization of LARP1, HCV core, and calnexin. Nuclei were counterstained with Hoechst 33342. LARP1 (red), core (magenta), and calnexin (green) were detected using Alexa-617, Alexa-594, and Alexa-488, respectively. Overlays were generated by the ImageJ software. Open and solid arrows show no or total colocalization, respectively. Bar = 5 μm. D. Statistical assessment of the colocalization of LARP1 and Calnexin. Red (LARP1) and green (Calnexin) fluorescence intensities were measured for each pixel along a 5-μm horizontal line centered around the arrow tips in (C) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data.
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pbio.1002421.g006: LARP1 relocalizes to the ER upon HCV infection.Cells were infected by HCV for 4 d prior to fixation, LARP1, NS5A, and calnexin staining, and confocal microscopy. A. Li diagrams and Li coefficient (B) calculations for LARP1 and Calnexin expression. Pixels present on the left and right sides of the y-axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the ImageJ software (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given single experiment (n = 3). C. Representative immunofluorescence-based localization of LARP1, HCV core, and calnexin. Nuclei were counterstained with Hoechst 33342. LARP1 (red), core (magenta), and calnexin (green) were detected using Alexa-617, Alexa-594, and Alexa-488, respectively. Overlays were generated by the ImageJ software. Open and solid arrows show no or total colocalization, respectively. Bar = 5 μm. D. Statistical assessment of the colocalization of LARP1 and Calnexin. Red (LARP1) and green (Calnexin) fluorescence intensities were measured for each pixel along a 5-μm horizontal line centered around the arrow tips in (C) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data.

Mentions: In this context, we verified whether HCV infection induced alterations in the expression pattern of LARP1 in infected cells. Since HCV NS5A appeared to bind to LARP1 with the highest affinity, we conducted immunofluorescence assays to confirm this finding. LARP1 signals were strongly reconfigured following HCV infection, adopted a granular pattern at the expense of their initial homogenous staining profile in naïve cells, and concentrated at the vicinity of lipid droplets visible as spheric structures surrounded by LARP1 and NS5A staining (Fig 5A). We confirmed that the HCV NS5A protein colocalized with LARP1 in infected cells using a plot profile assay (Fig 5B). This was further confirmed using calnexin, an independent ER marker. Indeed, LARP1 underwent general relocalization to ER-positive sites (i.e., relevant to translation of secreted proteins) in HCV+ cells, especially at the vicinity of classically core-decorated lipid droplets (Fig 6, zoomed insert). Li colocalization parameters presented in diagram Fig 6A and the coefficient (Fig 6B) between LARP1 and calnexin were significantly upregulated (>2.5-fold) following HCV infection (for more details on Li values, see S1 Text). Representative images and plot profiles (r = 0.16; p = 0.1 in naïve cells versus r = 0.38; p = 0.002 in HCV+ cells) of these colocalization levels are shown in Fig 6C and 6D, respectively. To determine whether LARP1 was localized in translationally active sites within infected cells, we compared LARP1 aggregation sites with puromycin(+) areas using the ribopuromycylation method [37]. Accordingly, LARP1 had significantly accumulated in the cytosol of HCV+ cells (Fig 7). Li diagrams (Fig 7A) and coefficient (Fig 7B) were significantly upregulated (1.3-fold) in HCV+ cells. Corresponding representative images (Fig 7C) and plot profiles (r = 0.31 in naïve cells versus r = 0.82 in HCV+ cells, Fig 7D) are shown. These data (i) show that LARP1 strongly relocates to ER-associated translationally active sites upon HCV infection, which comprise areas adjacent to lipid droplets, and (ii) led us to investigate whether the HCV-induced increase in Netrin-1 translation was, in turn, LARP1-mediated.


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)

LARP1 relocalizes to the ER upon HCV infection.Cells were infected by HCV for 4 d prior to fixation, LARP1, NS5A, and calnexin staining, and confocal microscopy. A. Li diagrams and Li coefficient (B) calculations for LARP1 and Calnexin expression. Pixels present on the left and right sides of the y-axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the ImageJ software (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given single experiment (n = 3). C. Representative immunofluorescence-based localization of LARP1, HCV core, and calnexin. Nuclei were counterstained with Hoechst 33342. LARP1 (red), core (magenta), and calnexin (green) were detected using Alexa-617, Alexa-594, and Alexa-488, respectively. Overlays were generated by the ImageJ software. Open and solid arrows show no or total colocalization, respectively. Bar = 5 μm. D. Statistical assessment of the colocalization of LARP1 and Calnexin. Red (LARP1) and green (Calnexin) fluorescence intensities were measured for each pixel along a 5-μm horizontal line centered around the arrow tips in (C) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data.
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Related In: Results  -  Collection

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pbio.1002421.g006: LARP1 relocalizes to the ER upon HCV infection.Cells were infected by HCV for 4 d prior to fixation, LARP1, NS5A, and calnexin staining, and confocal microscopy. A. Li diagrams and Li coefficient (B) calculations for LARP1 and Calnexin expression. Pixels present on the left and right sides of the y-axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the ImageJ software (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given single experiment (n = 3). C. Representative immunofluorescence-based localization of LARP1, HCV core, and calnexin. Nuclei were counterstained with Hoechst 33342. LARP1 (red), core (magenta), and calnexin (green) were detected using Alexa-617, Alexa-594, and Alexa-488, respectively. Overlays were generated by the ImageJ software. Open and solid arrows show no or total colocalization, respectively. Bar = 5 μm. D. Statistical assessment of the colocalization of LARP1 and Calnexin. Red (LARP1) and green (Calnexin) fluorescence intensities were measured for each pixel along a 5-μm horizontal line centered around the arrow tips in (C) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data.
Mentions: In this context, we verified whether HCV infection induced alterations in the expression pattern of LARP1 in infected cells. Since HCV NS5A appeared to bind to LARP1 with the highest affinity, we conducted immunofluorescence assays to confirm this finding. LARP1 signals were strongly reconfigured following HCV infection, adopted a granular pattern at the expense of their initial homogenous staining profile in naïve cells, and concentrated at the vicinity of lipid droplets visible as spheric structures surrounded by LARP1 and NS5A staining (Fig 5A). We confirmed that the HCV NS5A protein colocalized with LARP1 in infected cells using a plot profile assay (Fig 5B). This was further confirmed using calnexin, an independent ER marker. Indeed, LARP1 underwent general relocalization to ER-positive sites (i.e., relevant to translation of secreted proteins) in HCV+ cells, especially at the vicinity of classically core-decorated lipid droplets (Fig 6, zoomed insert). Li colocalization parameters presented in diagram Fig 6A and the coefficient (Fig 6B) between LARP1 and calnexin were significantly upregulated (>2.5-fold) following HCV infection (for more details on Li values, see S1 Text). Representative images and plot profiles (r = 0.16; p = 0.1 in naïve cells versus r = 0.38; p = 0.002 in HCV+ cells) of these colocalization levels are shown in Fig 6C and 6D, respectively. To determine whether LARP1 was localized in translationally active sites within infected cells, we compared LARP1 aggregation sites with puromycin(+) areas using the ribopuromycylation method [37]. Accordingly, LARP1 had significantly accumulated in the cytosol of HCV+ cells (Fig 7). Li diagrams (Fig 7A) and coefficient (Fig 7B) were significantly upregulated (1.3-fold) in HCV+ cells. Corresponding representative images (Fig 7C) and plot profiles (r = 0.31 in naïve cells versus r = 0.82 in HCV+ cells, Fig 7D) are shown. These data (i) show that LARP1 strongly relocates to ER-associated translationally active sites upon HCV infection, which comprise areas adjacent to lipid droplets, and (ii) led us to investigate whether the HCV-induced increase in Netrin-1 translation was, in turn, LARP1-mediated.

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