Figure 1.
Cytoplasmic localization of Nups in HCV-infected tissue culture cells.
Huh7.5 cells were uninfected or infected with HCV for four days. A) Localization of Nups in cells either uninfected (Un) or HCV-infected (HCV) was evaluated by indirect immunofluorescence microscopy using antibodies specific for the indicated Nups or lamin B (green) and HCV core (red). DNA was stained with DAPI (blue). Scale bar, 2 µm (A). B) Changes in the cytoplasmic levels of Nups induced by HCV infection were quantified. The ratio of cytoplasmic to nuclear fluorescence levels were calculated for the indicated Nups and lamin B in uninfected and HCV-infected cells (n≥10). The statistical significance of differences between uninfected and infected ratios was determined (p-values less than 0.05 (*), 0.01 (**) and 0.001 (***) are indicated). C) Total cell lysates were isolated from uninfected (UN) or infected (HCV) Huh7.5 cells and subjected to subcellular fractionation. Western blotting using antibodies specific for the indicated proteins was used to evaluate the protein levels in each of the indicated subcellular fractions. In addition to the Nups and HCV proteins indicated, markers for residue ER (calnexin), NE (lamin B), mitochondria (VDAC), and MAM (FacL4) proteins are shown. Equal amounts of total protein were loaded into each lane. All samples were run on the same gel and images shown are derived from scans of the same membrane.
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.
Figure 3.
HCV infection increases RNA and protein levels of a subset of Nups.
A) Total RNA was isolated from cell lysates at the indicated time points after infection of Huh7.5 cells with HCV, and levels of specific mRNA transcripts were assessed by qPCR. Values for each sample were normalized to HPRT and are expressed relative to uninfected cells (day 0 time point). HCV RNA levels are shown as fold change relative to HPRT. Error bars indicate standard error (based on ≥3 experiments) and statistical significance was evaluated using t-tests comparing each infected sample to an uninfected control sample. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated. B) Cell lysates were harvested from HCV infected Huh7.5 cells four days after infection and Nup proteins levels were determined by western blotting using antibodies specific for Nup98, Nup153, Nup155, and Nup358. Protein levels were quantified and normalized to tubulin levels and the fold-change is relative to uninfected cells. Error bars were determined using data from ≥3 experiments.
Figure 4.
Identification of Kaps that physically interact with HCV proteins through NLS sequences.
A) Constructs encoding the indicated V5-tagged HCV protein or an empty V5 vector (Ctrl) were transfected into HEK293T cells. 12 hrs after transfection, cells were subjected to no treatment, treatment with Kap β3/IPO5-NLS peptides, treatment with control penetratin peptides, or transfection with a construct encoding for a cNLS-GFP fusion protein and incubated for an additional 36 hrs. Following treatment, cells were lysed and V5-tagged HCV proteins were immunoprecipitated with anti-V5 antibodies (IP). Associated proteins were detected by western blotting using antibodies against the indicated Nup or Kap (WB). B) The subcellular localization of Kap β3/IPO5 in Huh7.5 cells uninfected or infected with HCV was determined by indirect immunofluorescence microscopy using antibodies specific to Kap β3/IPO5 (green) and HCV core (red). DNA was stained with DAPI (blue). Scale bars, 5 µm.
Figure 5.
Depletion of Nups and Kaps inhibits HCV replication.
A–D) Huh7.5 cells were coinfected with HCV and lentivirus encoding shRNAs directed against Nup98, Nup155, Nup153, Kap β3/IPO5, NDC1, or a scrambled control sequence for four days. The effects of Nup or Kap depletion on HCV titers were evaluated by qPCR analysis of HCV RNA levels in cell extracts (panel A) or in the culture supernatant (panel B) from HCV infected Huh7.5 cells co-infected with and without lentivirus. In addition, intracellular levels of the HCV core protein were examined by quantitative western blotting using antibodies specific for HCV core (panel C). Values for each sample are normalized to HPRT mRNA (Panel A and B) or α-tubulin (Panel C) and are expressed as fold-change relative to HCV infected cells not treated with lentivirus. Error bars indicate standard error (based on ≥3 experiments) and statistics are based on t-tests comparing each Nup or Kap specific shRNA treated sample to samples expressing the scrambled shRNA control. D) Huh7.5 cells were grown as described in panel A and the infectious titers of HCV particles present in the media of cells depleted of the indicated proteins were determined. Focus-forming units were determined using indirect immunofluorescence microscopy. Values shown represent focus-forming units per mL of medium (FFU/mL). E–F) Huh7.5 cells were infected with HCV and 4 hrs post infection a penetratin peptide, a Kap β3-NLS peptide, or a penetratin peptide containing a N-terminal Kap α NLS (Kap α-NLS) was added to the media. Four days later the effects of these peptides on HCV RNA levels in intracellular (panel E) and extracellular (panel F) compartments were assessed by qPCR analysis. Values for each sample are normalized to HPRT mRNA levels and expressed as fold change relative to cells receiving no peptide. Error bars indicate standard error (based on ≥3 experiments) and statistics based on t-tests comparing cells treated with penetratin alone to those treated with the Kap β3-NLS peptide or the Kap α-NLS containing peptide. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated.
Figure 6.
cNLS-GFP reporter protein and Ran accumulated in the vicinity of HCV replication/assembly.
A) Uninfected or HCV-infected Huh7.5 cells were transfected with a construct encoding a chimeric protein consisting of an N-terminal cNLS sequence followed by two tandemly repeated GFP molecules 2 days after infection. On day 4 after HCV infection, the cNLS-GFP reporter was visualized by fluorescence microscopy (green) and its location compared to HCV NS5A (red) detected by immunofluorescence microscopy. DNA was detected with DAPI (blue). All cell producing the cNLS-GFP reporter exhibited a nuclear signal. Visible levels of the cNLS-GFP reporter were also detected in the cytoplasm of ∼81% of infected cells (n = 193) and ∼23% of uninfected cells (n = 198). Scale bars, 5 µm. B) Localization of Ran in Huh7.5 cells, either uninfected or infected with HCV for four days, was evaluated by indirect immunofluorescence microscopy using antibodies specific for Ran (green) and HCV NS5A (red). DNA was stained with DAPI (blue). Scale bar, 5 µm. C) Cytoplasmic and nuclear fluorescence signal levels produced by the cNLS-GFP reporter protein or the Ran specific antibodies were used to determine cytoplasmic to nuclear fluorescence ratios in uninfected and HCV infected cells. Fluorescence levels were calculated using ImageJ software and the statistical significance of differences in the ratios detected in uninfected versus HCV-infected cells was evaluated using t-tests. Asterisks (***) denotes a p-value of less than 0.001.
Figure 7.
Model for the function of cytoplasmic NPCs in HCV infection.
In HCV infected cells, cytoplasmically positioned NPCs are predicted to form channels across double membrane structures of the membranous web. These NPCs are proposed to facilitate movement of NLS-containing proteins, such as HCV core, NS2, NS3, NS5A and host nuclear proteins, from the surrounding cytoplasm across double membrane structures of the membranous web while excluding proteins lacking NLS sequences, such as pattern recognition receptors (PRRs), from regions of HCV replication and assembly events. We also speculate that Nups (such as Nup155 and Nup153) may be recruited to regions of the membranous web where HCV replication and virion assembly would employ the ability of these Nups to contour membranes.