Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication
Figure 2
Identification of Nups that physically interact with HCV proteins.
A) Cell lysates were isolated from uninfected (Un) and HCV-infected (HCV) Huh7.5 cells four days after infection. The nuclear fraction was removed from the lysates by sedimentation and the remaining cytoplasmic fraction was incubated with the monoclonal antibody mAb414 specific for a subset of Nups. Proteins present in cell lysates and mAb414 immunoprecipitates (mAb414 IP) were analyzed by western blotting using antibodies specific for HCV. B) Constructs encoding for the indicated V5-tagged HCV proteins or an empty V5 vector (ctrl) were transfected into HEK293T cells and expressed for 48 hrs. V5-tagged proteins were immunoprecipitated with anti-V5 antibodies and associated proteins were evaluated by western blotting using antibodies against the indicated Nups and the V5 epitope. C) Subcellular localization of Nup155 in Huh7.5 cells expressing the indicated V5-tagged HCV proteins was examined by indirect immunofluorescence microscopy using antibodies directed against Nup155 (green) and the V5 epitope (red). DNA is visualized with DAPI (blue) and arrows point to transfected cells. Pearson's colocalization coefficients were calculated to assess colocalization of Nup155 and the indicated V5 tagged protein and are shown at the top of the merged panel. Values >0.5 are considered significant. Scale bar, 10 µm.