Fig 6. Clathrin-dependent endocytosis is involved in RHDV internalization and is regulated by NCL.

(A) Clathrin-dependent endocytosis is involved in RHDV internalization. RK-13 cells of three groups were pretreated with CPZ (30 mM), EIPA (50 mM), or Nys (100 mM) for 2 h. Another group of cells was transfected with NCL siRNA (100 nM) for 24 h. Cells were then prompted to internalize mRHDV (MOI = 1) and harvested for RNA analysis. The percentage of RHDV VP60 copies was calculated as a ratio to the value obtained from untreated RK-13 cells. DMSO and non-specific siRNA were used as negative controls. The data are presented as the mean and standard deviation (error bars) of three samples per group (**p < 0.01). All experiments were repeated at least three times and showed consistent results. (B) mRHDV-FITC (MOI = 1) infected RK-13 cells, treated with procedures described above, were subjected to the internalization assay and harvested for flow cytometry analysis. (C) RK-13 cells of three groups were pretreated with CPZ (30 mM), EIPA (50 mM), or Nys (100 mM) for 2 h. Another group cells was transfected with NCL siRNA (100 nM) for 24 h. Then, the cells were used for the internalization assay with mRHDV-FITC (MOI = 1) and subsequent immunofluorescence analysis. The RHDV virion is green, cell nucleus is blue. DMSO and control siRNA were employed as negative controls.