FIG. 7.

HCV RCs are associated with MTs and the ER. Huh-7.5-I/5A-GFP-6 cells were fixed and processed for indirect immunofluorescence staining as described in Materials and Methods. Primary mouse MAbs against α-tubulin or calnexin were used to detect MTs and the ER, respectively. Stacks of images were acquired by confocal laser scanning microscopy and deconvoluted. (A) Association of HCV RCs with MTs. An overview image is shown at the top. MTs (red) form a dense network in Huh-7.5 cells. NS5A-GFP (green) is found in large and small structures. The area marked by the dashed box is enlarged below and shows that the structures are located in close proximity to MTs. Green intensity settings were adjusted in the enlargement to match the ER signal in this area. (B) Association of HCV RCs with the ER. An enlarged region from a deconvoluted image stack is shown (see Fig. 8C for an overview image). Small NS5A-GFP structures (green) are closely associated with the ER (red) and are found either on top or directly adjacent to ER tubules; however, colocalization on a per pixel basis is hardly detectable. NS5A-GFP structures often bridge a gap in the ER marker signal (arrow), which suggests that they may exclude the ER marker protein. Scale bars indicate reference distances of 20 and 1 μm, respectively.