Figure 8. The N-terminal part of NS4B assumes a dual topology in a replicative context.

(A) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see Materials and Methods section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the Materials and Methods section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (B) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software [70]. (C) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 [15]. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).