FIG. 4.

Morphology of cell lines with an sfl genome and localization of viral proteins. (A) Ultrathin section obtained from cell line 21-5 after glutaraldehyde-osmium fixation and ERL embedding. Magnification, ×28,500. The cells exhibit numerous lipid vesicles (V) of different sizes surrounded to different degrees by electron-dense rims resembling a thickened membrane. (B) Ultrathin section of parental Huh-7 cells (fixation, embedding, and magnification as for panel A). Note the absence of the electron-dense rim around the border of the vesicles. (C to E) Localization of HCV E2 (C) and core (D) proteins and the Golgi-resident protein Golgin 97 (E) in cell line 21-5 by indirect immunofluorescence. Representative sections are shown at a magnification of ×304. Bar, 50 μm. Nuclei were counterstained using bisbenzimide (Hoechst). Note the granular staining pattern of the core protein that to some extent resembles the distribution of the Golgi marker Golgin 97. (I and J) Specificity control for the anti-core and anti-E2 sera. Cells harboring a subgenomic replicon were fixed and stained as cells with the sfl genome. Magnification, ×285. (F to H) Localization of E2, core, and Golgin 97 in cell line 21-5 treated with 20 nM brefeldin A for 3 h. Detection of Golgin 97 is abrogated (H), while the distribution of E2 and core remains essentially unchanged (F and G, respectively). Magnification, ×304.