FIG. 3.

Intracellular infectious HCV precursors are susceptible to nonproteasomal degradation. Persistently infected cells were treated with the proteasome inhibitors lactacystin, MG132, and ALLN (40 μM) at 37°C. (A) Inhibition of the proteasome was demonstrated by the accumulation of ubiquitinated proteins, as shown by Western blotting with anti-ubiquitin antibodies of total cell extracts obtained after a 16-h incubation period. Samples were analyzed for GAPDH content as a loading control. Molecular mass markers are shown in kilodaltons. (B) Intracellular infectivity (FFU/ml) was determined at 6 h posttreatment in the presence of lactacystin, MG132, ALLN (40 μM), or ALLN (40 μM) in combination with BFA (1 μg/ml). (C) Time course showing rapid intracellular infectious particle accumulation in the presence of ALLN (40 μM) (white bars) compared to that in the control (black bars). (D) Extracellular infectivity after treatment of persistently infected cells with 40 μM ALLN for 9 h at 37°C. Extracellular infectivity titers were determined in the presence (white bars) and absence (black bars) of the drug, as described in Materials and Methods, and are presented as averages and standard deviations for triplicate experiments (n = 3).