Immunogold labeling. For immunogold EM localization of HCV proteins, cells were fixed (0.1% glutaraldehyde, 4% paraformaldehyde in phosphate buffered saline) and embedded in LR Gold resin (Ted Pella) using techniques described by Berryman and Rodewald [52] . Antibodies were pre-absorbed with Huh7 cell homogenate for 1-24 hrs. Thin sections on grids were blocked with 3% bovine serum albumin in TBS (Tris buffered saline) for 1 hr at RT, incubated with primary antibodies for 1 hr at RT, followed by gold-labeled goat anti-mouse IgG or goat antirabbit IgG for 1 hr at RT. The antibody complexes were stabilized with 2% glutaraldehyde in water, and the sections were stained with osmium vapor and lead citrate and examined as described above [53, 54] . Huh7 and Core-off cells were used as negative controls for antibody bindings. Multiple non-overlapping areas were scored for the frequency of HCV antigen localization and for the abundance of the antigen in each area. A positive signal is defined as the presence of at least two gold particles in a given organelle.