Core-NS5A complexes are transported from perinuclear region to the plasma membrane via microtubules.

<p>The HCV-infected cells (at day 10 p.i.) were labeled with antibodies to Core protein (red) (A, B), NS5A (blue, A; green, B) (upper rows), NS4B (blue, A; green, B) (lower row) or tubulin (green) (A). Plasma membrane and nuclei were stained with WGA Alexa Fluor 647 conjugate (blue) (B) and DAPI (gray), respectively. The second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (A). Colocalization of Core with NS5A or NS4B is depicted as magenta (A) or yellow (B). Colocalization of Core-NS5A or -NS4B protein complexes with microtubules (A) or plasma membrane (B) is depicted as white. The microtubule end (white arrow) is closest to the cell periphery (A). (B) At the right is an enlarged area, marked with a white box, from the merged image. Colocalization of NS5A with Core protein was observed in the membrane curvature (white arrow). PM, plasma membrane; Bars, 10 µm. (C) Quantitation of antibody-labeled signals of viral proteins at the plasma membrane. Images from 20 cells were counted manually using an original magnification of ×630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (D, E) NS5A and Core are localized in clusters or patches on the plasma membrane. In parallel, the HCV-infected cells were labeled with anti-NS5A (D) or anti-Core (E) antibodies. Bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. Sections were visualized by EM. Arrows, gold-labeled NS5A (D) or Core protein (E). (F) Immuno-EM of Core-NS5A complex colocalized at the plasma membrane. The HCV-infected cells were co-labeled with antibodies against NS5A (6 nm) and Core (18 nm). Shown is a view of the plasma membrane. Arrowhead, gold-labeled NS5A. Arrow, gold-labeled Core. PM, plasma membrane; Bars, 100 nm (D, F) and 200 nm (E).