Role of Ras-related Nuclear Protein/Polypyrimidine Tract Binding Protein in Facilitating the Replication of Hepatitis C Virus

Figures

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  Fig. 1. HCV infection induces the expression of RAN. Huh7.5.1 cells were infected with the JC1 virus. Naïve Huh7.5.1 cells were used as a MOCK-infected control. (A) The replication of HCV was examined using the luciferase assay. (B) Intracellular core and RAN levels were analyzed using western blotting at 48 h post-infection. (C) Intracellular RAN levels were determined using quantitative reverse transcription-PCR at 48 h post-infection. (D) The ratios of core/GAPDH and RAN/GAPDH are shown. Representative images of three independent experiments are presented. *p<0.01, compared with control cells.

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  Fig. 2. HCV infection induces the nucleo-cytoplasmic shuttling of PTB. (A and B) The cytoplasm to nuclear ratio for PTB expression levels was detected using western blotting. C, cytoplasm; N, nucleus; PCNA, proliferating cell nuclear Ag. The representative blots of three independent experiments are shown. (C) Imaging of PTB fluorescence intensity. Cellular PTB was stained with anti-PTB mouse antibody and then stained with Alexa fluor 488 anti-rabbit secondary antibody (green color). The nucleus was stained with DAPI (blue color). Merge indicates the combination of PTB fluorescence intensity with nuclear fluorescence intensity. *p<0.01, compared with control cells.

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  Fig. 3. Silencing effects of siRNAs on the mRNA and protein expression levels of RAN. Huh7.5.1 cells were treated with siRNAs specific to RAN (003, 004 and 005); an irrelevant siRNA (NC) was used as a control in each experiment. (A–B) The expression levels of RAN after siRNA treatment were determined by quantitative reverse transcription-PCR and western blotting. GAPDH mRNA and protein were used for normalization. (C) The ratio of RAN/GAPDH is shown. The results represent three independent experiments. *p<0.01, compared with MOCK-treated cells.

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  Fig. 4. Knockdown of RAN inhibits HCV replication and the cytoplasmic redistribution of PTB. (A–C) Inhibition of HCV replication was investigated using the luciferase assay, quantitative reverse transcription-PCR and western blotting. Naïve Huh7.5.1 cells were used as a control. (D–E) Inhibition of the cytoplasmic redistribution of PTB was studied using western blotting. *p<0.01, compared with control cells. **p<0.01, compared with JC1 virus-infected cells.

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  Fig. 5. Colocalization of RAN with PTB on Huh7.5.1 cells infected with JC1 virus. Cells infected with JC1 virus were fixed with ice-cold methanol at 48 h post-infection. The fixed cells were incubated with anti-RAN antibody or anti-PTB antibody followed by a secondary antibody labeled with Alexa fluor 647 (red, RAN) or Alexa fluor 488 (green, PTB). The colocalized signal (yellow) was visualized by merging the two images. All the experiments were performed in triplicate.

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  Fig. 6. RAN physically interacts with PTB in Huh7.5.1 cells. Cells were extracted and immunoprecipitated with the anti-RAN antibody, and the immunocomplexes were analyzed by western blotting using the anti-PTB antibody. The IgG antibody was used as a negative control.