Fig 2: HCV Core is targeted to lipid droplets even in the ESCRT knockdown cells.The RSc cells transduced with a control lentiviral vector (Con), the TSG101 knockdown (TSG101i), the Alix knockdown (Alixi), the Vps4B knockdown (Vps4Bi), or the CHMP4b knockdown (CHMP4bi) cells were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy. Cells were stained with anti-HCV Core (CP-9 and CP-11 mixture) and were then visualized with Cy3 (red). Lipid droplets and nuclei were stained with BODIPY 493/503 (green) and DAPI (blue), respectively. Images were visualized using confocal laser scanning microscopy. Colocalization is shown in yellow (Merged).

Fig 3: ESCRT components are required for the infectious HCV production.(A) Inhibition of TSG101, Alix, Vps4B, or CHMP4b protein expression by shRNA-producing lentiviral vectors. The results of the Western blot analysis of cellular lysates with anti-TSG101, anti-Alix, anti-Vps4B, anti-CHMP4b, anti-Chk2, anti-HSP70, or anti-β-actin antibody in RSc cells expressing shRNA targeted to TSG101 (TSG101i), Alix (Alixi), Vps4B (Vps4Bi), or CHMP4b (CHMP4bi) as well as in RSc cells transduced with a control lentiviral vector (Con) are shown. Real-time LightCycler RT-PCR for TSG101 (B), Alix (C), Vps4B (D), or CHMP4b mRNA (E) was performed as well as for β-actin mRNA in triplicate. Each mRNA level was calculated relative to the level in RSc cells transduced with a control lentiviral vector (Con) which was assigned as 100%. Error bars in this panel and other figures indicate standard deviations. (F) MTT assay of each knockdown RSc cells at the indicated time. (G) The levels of HCV Core in the culture supernatants from the stable knockdown RSc cells 97 h after inoculation of HCV-JFH1 at an MOI of 0.1 were determined by ELISA. Experiments were done in triplicate and columns represent the mean Core protein levels. (H) The infectivity of HCV in the culture supernatants from the stable knockdown RSc cells 97 hrs after inoculation of HCV-JFH1 at an MOI of 0.1 was determined by a focus-forming assay at 48 hrs post-infection. Experiments were done in triplicate and each virus titer was calculated relative to the level in RSc cells transduced with a control lentiviral vector (Con) which was assigned as 100%. Asterisks indicate significant differences compared to the control treatment. *P<0.05; **P<0.01. (I) The level of intracellular genome-length HCV-JFH1 RNA in the cells at 97 hrs post-infection was monitored by real-time LightCycler RT-PCR. Results from three independent experiments are shown. (J) Inhibition of TSG101, Alix, Vps4B, or CHMP4b protein expression by 72 hrs after transient transfection of RSc cells with a pool of control siRNA (Con) or a pool of siRNA specific for Alix, Vps4B, or CHMP4b (50 nM). The results of the Western blot analysis of cellular lysates with anti-TSG101, anti-Alix, anti-Vps4B, anti-CHMP4b, or anti-β-actin antibody is shown. (K) MTT assay of each knockdown RSc cells at the indicated time. (L) The levels of HCV Core in the culture supernatants were determined by ELISA 24 hrs after inoculation of HCV-JFH1. RSc cells were transiently transfected with a pool of control siRNA (Con) or a pool of siRNA specific for TSG101, Alix, Vps4B, or CHMP4b (50 nM). At 48 hrs after transfection, the cells were inoculated with HCV-JFH1 at an MOI of 5 and incubated for 2 hrs. Then, culture medium was changed and incubated for 22 hrs. Experiments were done in triplicate and each Core level was calculated relative to the level in the culture supernatants from the control cells and indicated below. (M) The infectivity of HCV in the culture supernatants from the transient knockdown RSc cells 24 hrs after inoculation of HCV-JFH1 at an MOI of 5 was determined by a focus-forming assay at 48 hrs post-infection. Experiments were done in triplicate and each virus titer was calculated relative to the level in RSc cells transfected with a control siRNA (Con) which was assigned as 100%. Asterisks indicate significant differences compared to the control treatment. *P<0.05; **P<0.01.