FIG. 2.
Small NS5A-GFP structures contain further HCV replicase components. Huh-7.5-I/5A-GFP-6 cells were fixed, processed for indirect immunofluorescence staining, and analyzed by confocal laser scanning microscopy as described in Materials and Methods. Viral proteins were labeled with the primary mouse MAb 9E10 anti-NS5A or 7019 anti-NS3, which were detected with an Alexa Fluor 546-labeled goat anti-mouse antibody (Alx546). (A) Colocalization between NS5A-GFP autofluorescence (green) and 9E10 anti-NS5A staining (red). Both NS5A specific signals colocalize well, which is also indicated by the high Pearson correlation coefficient (Rc = 0.90). (B) Colocalization between NS5A-GFP (green) and NS3 (red). NS5A and NS3 also colocalize well (Rc = 0.79). (C and D) Enlargement of the dashed box region in panel A (C) and enlargement of the dashed box region in panel C (D) focus on several small structures. Points a and b, as well as points c and d, define two line segments that each cross several structures. Intensity profiles along the line segments, shown on the right of the images, demonstrate that NS5-GFP and NS3 also colocalize in small structures. Scale bars indicate reference distances of 5 and 1 μm, respectively; the length of the line segments between points a and b, as well as points c and d, are depicted underneath the intensity profiles. Intensity adjustments are reflected by the intensity scale bars as detailed in Materials and Methods.