Figure 9. Subcellular localization of NS2 in assembly deficient mutants in NS5A protein.

(A, B) Phenotype of the NS5A mutants. Virus infectivity (A), extra (black bars) and intracellular (light grey bars) core determination (B) were performed as described in Figure 2. The following viruses were analyzed: JFH-HA (WT), JFH-S/A-HA (S/A), JFH-3BS/A-HA (3BS/A) and JFH-S/D-HA (S/D). In addition, JFH-HA-PP (PP) was also analyzed in parallel. (C) Analysis of the expression and phosphorylation of the NS5A mutants. The hyperphosphorylated form of NS5A is indicated by an asterisk. The presence of core and NS5A was confirmed by Western blotting and the actin content was also analyzed to verify that equal amounts of cell lysates have been loaded. (D) Effect of mutations on the accumulation of NS2 in dotted structures. Huh-7 cells electroporated with JFH-HA RNA (WT) or mutant genomes were grown on coverslips and fixed at 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red) and anti-NS5A (green) antibodies. The nuclei were stained with DAPI. Representative confocal images of NS2 and NS5A labelings are shown in grey, and the merge images in color. Bar, 10 µm. (E) Mutations in NS5A protein drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA or the indicated mutants were grown on coverslips, fixed at 72 h post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells (at least 228). Error bars indicate SD from at least two independent experiments.