Figure 4. NS2 colocalization with viral proteins in association with LDs.

JFH-HA electroporated cells grown on coverslips were fixed at 72h post-electroporation and processed for triple-label immunofluorescence for HA-NS2 (red), the LDs (green), and (A) NS5A or (B) core protein (C) (blue). Individual confocal images of each labeling are shown in left panels, with a merged image of the three channels in the top left panel. Two by two overlays are shown in right panels. A view of the entire cell is shown in the top right panel. Bar, 10 µm. (C) The kinetics of virus production for JFH-HA. Huh-7 cells were electroporated with viral RNA transcribed from JFH-HA construct. At different time points post-electroporation, the virus infectivity present in the supernatants was determined by titration of foci forming units (FFUs)(upper panel). Error bars indicate SE from at least two independent experiments performed in duplicate. The kinetics of NS2/NS5A positive dots parallels virus production (lower panel). Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at different time points post-electroporation and labeled with anti-HA and anti-NS5A antibodies. The cells which presented at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted infected cells. At least 300 infected cells were counted for each time point. Error bars indicate SE from at least two independent experiments. (D) NS2 detection by immuno-EM microscopy. JFH-HA electroporated cells grown in 75 cm² flasks were fixed at 72h post-electroporation and processed for immuno-electron microscopy labeling with anti-HA antibody. A representative image is shown where three HA-NS2 clusters (arrows) could be observed in the proximity of a LD. Two of these clusters of gold particles (white arrows) are lying on well-preserved ER bilayers. For one of these clusters of gold particles, a connection between the ER bilayer and the LD could be observed (white arrowhead). A third cluster of gold particles (black arrow) is observed on a less preserved ER bilayer.