Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication
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.