Figure 6

Induction of γ-H2AX in HCV replicating or vmr11 transfected cells. (A) γ-H2AX is induced by vmr11 in HCV infected cells: (from left to right), Human hepatocyte cultures were either mock transfected (Control), or transfected with 1 μg HCV1a (H77) genomic RNA, or with 50 nM vmiR11 “mimic” oligonucleotides. As controls, HCV1a genomic RNA or vmr11-transfected cultures were treated with 50 nM vmr11 antagomir (“LNA”). The cells were harvested 3 days post-transfection and analyzed by Western blot for γ-H2AX. The numbers beneath indicate relative values of γ-H2AX normalized to β-Actin loading control. (B) Induction of γ-H2AX correlates with vmr11 mutants that inhibit nuclear PTEN protein: Western blots (of cells transfected with vmr11 oligonucleotides, similar to the results shown in panel A) were compared with changes in γ-H2AX levels in cells transfected with either the WT vmr11, or with one of the vmr11 mutants (shown in Figure 5A). The numbers underneath represent relative values of γ-H2AX protein normalized to β-Actin loading control (C) Immunofluorescence staining of γ-H2AX (FITC) in HCV replicating or vmr11 transfected cells: Human hepatocytes were either transfected with HCV1a (1 μg viral genomic RNA; HCV replication is marked with NS5A viral antigen (Texas red stain), or transfected with vmr11 (50 nM) oligonucleotides, and processed for immunofluorescence viewing after 48 hours. γ-H2AX was stained with FITC and NS5A with Texas Red. Stained cell foci shown are at magnification 20x and 100x (inset). (D) Mechanism of regulation of nuclear PTEN deficiency by HCV: Schematic of nuclear PTEN restriction in HCV-infected cells that is mediated by translational silencing of Transportin-2 with vmr11.