FIGURE 6.

VP2 reduces ORAOV1. (A–D) The ORAOV1 expression is reduced by VP2. Hela cells in 6-well pate were transfected with the indicated amounts of pEGFP-N1-vp2 or pEGFP-N1 controls. Total amounts of plasmid DNA were equalized to 500 ng using pRK5-flag. Forty eight hours after transfection, cell lysates were examined with Western Blot (A). The densities of bands in A were quantitated by densitometry. The relative levels of ORAOV1 were calculated as follows: band density of ORAOV1/ that of β-actin (B). (C) DF-1 cells were mock infected or infected with IBDV at different MOI of 0.5, 5, or 50. Mock infected cells were treated in the same way as IBDV infection at the indicated MOI but using culture medium instead of the virus 24 h after infection, the lysates of IBDV-infected cells were examined with Western Blot. The densities of bands in (C) were quantitated by densitometry as described above (D). Data are representative of three independent experiments, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (E) The reduction of ORAOV1 correlated to the enhanced apoptosis during IBDV infection. Cell samples in (C) were collected and stained with PE annexin-V for apoptosis analysis by flow cytometry. (F) Examination of oraov1 mRNA levels in VP2 transfected cells. Hela cells were transfected with the 500 ng pEGFP-N1-vp2 or pEGFP-N1 controls. The cell samples were collected for qRT-PCR assay 48 h post transfection. (G) Examination of oraov1 mRNA levels in IBDV infected cells. DF-1 cells were mock infected or infected with IBDV at an MOI of 5. Twenty four hours after infection, the cell samples were collected for qRT-PCR assay. The expression levels of mRNA were calculated in relation to that of GAPDH. Results are representative of three independent experiments.