Silencing of GBP1 affects lysosomal functionality and leads to a reduction in specific infectivity of HCV viral particles.

(A) Z-stack and 3D-reconstruction (3D inset xy-section, 3D inset) of stably HCV replicating Huh7.5 cells (scr RNA) to visualize LAMP2 structures colocalizing with NS3 and GBP1 as part of the RCs, indicated by the white arrows. GBP1 (red), NS3 (green) and LAMP2 (cyan) were detected using specific antisera. Nuclei were visualized with DAPI. Scale bar, 10 μm. Images are representative of 3 biological replicates. (B) Z-stack and 3D-reconstruction (inset) of HCV-replicating Huh7.5 cells 72 h after GBP1 silencing to visualize HCV core stacking outside and NS3 inside lysosomal structures, as indicated by the white arrows. GBP1 (red), HCV core/ NS3 (green) and LAMP2 (cyan) were detected using specific antisera. Nuclei were visualized with DAPI. Scale bar, 10 μm. Images are representative of 3 biological replicates. (C) CLSM analysis of stably HCV replicating Huh7.5 cells (Jc1) 72 h after GBP1 silencing. LysoTracker Deep Red staining (magenta) was performed for 1h at 37°C before fixing the cells with 4% FA. GBP1 (red), HCV core (green) and LAMP2 (cyan) were detected using specific antisera. Nuclei were visualized with DAPI. Scale bar, 50 μm (inset 10 μm). Quantification of the relative CTCF (corrected total cell fluorescence) of LAMP2 and LysoTracker. For each condition, a minimum of 10 cells was analyzed. To detect the impact of GBP1 silencing on lysosomal acidification the ratio LAMP2/Lysotracker was calculated. Images are representative of 3 independent experiments. All immunofluorescence images were deconvoluted using the LasX Lightning Tool using the adaptive algorithm. (D) Detection of intra- and extracellular viral titers by TCID50 72 h after GBP1 silencing. N = 3 (intracellular), 9 (extracellular) biological replicates. (E) Detection of extracellular viral genomes by qPCR 72 h after GBP1 silencing. Relative change of the extracellular HCV genomes were referred to the untreated (UT) scrambled (scr) control (set to 1). N = 6 biological replicates. (F) The specific infectivity after GBP1 silencing was determined by calculating the ratio of the number of extracellular infectious viral particles (TCID50/ml), measured by TCID50, and the total amount of extracellular viral genomes (genomes/ml) determined by qPCR. (G) CLSM analysis of stably HCV replicating Huh7.5 cells (Jc1) 72 h after GBP1 silencing. To induce autophay the cells were treated with Rapamycin (100 nM) 16 h before harvesting. LysoTracker Deep Red staining (magenta) was performed for 1h at 37°C before fixing the cells with 4% FA. GBP1 (red), HCV core (green) and LAMP2 (cyan) were detected using specific antisera. Nuclei were visualized with DAPI. Scale bar, 50 μm (inset 10 μm). Quantification of the relative CTCF (corrected total cell fluorescence) of LAMP2 and LysoTracker. For each condition, a minimum of 4 cells was analyzed. To detect the impact of GBP1 silencing on lysosomal acidification the ratio LAMP2/Lysotracker was calculated. All inset immunofluorescence images were deconvoluted using the LasX Lightning Tool using the adaptive algorithm. For all experiments, statistics were performed as mean ± SEM, one-way ANOVA with *p<0.05, **p<0.01, ****p<0.0001, ns = not significant.