FIG 3.

pAPN promotes PDCoV internalization. (A) Scheme of the PDCoV entry assay. The same quantities of HEK293 and HEK293-APN cells were plated into 10-cm² dishes, and after the cells had adhered, they were infected with PDCoV at an MOI of 100 for 2 h at 37°C. The cells were then harvested, treated with pronase (7 mg/ml), washed three times with ice-cold PBS, and centrifuged at 500 × g for 7 min at 4°C. The cell pellet was treated with hypotonic lysis buffer, lysed with a Dounce homogenizer, and centrifuged at 1,000 × g for 4 min at 4°C to remove the nuclear pellet. The supernatant fraction was centrifuged at 144,000 × g for 2 h at 4°C in a 50% sucrose solution in PBS. (B) The pellet fraction was analyzed by Western blotting (WB). The protein bands were then quantified by densitometry relative to the levels of N protein. This experiment was performed three times. A representative result is shown. (C) Knockdown of pAPN in IPI-21 cells. IPI-21 cells were transfected with either the indicated pAPN-specific siRNAs (100 nM siRNA-1 to −4) or 100 nM scrambled siRNA control (nontargeting control [NC]). At 24 h posttransfection (hpt), pAPN was quantified by Western blotting. This experiment was performed three times, and a representative result is shown. (D) Knockdown of pAPN reduced PDCoV replication in IPI-21 cells. IPI-21 cells were transfected with the indicated pAPN-specific siRNAs (100 nM siRNA-1 to -4) or 100 nM scrambled siRNA control (siScr) and then infected with PDCoV at an MOI of 100 for 2 h at 37°C, as described above. The protein bands then were quantified by densitometry relative to the levels of N protein. This experiment was performed three times. A representative result is shown. (E) Upon reaching 80% confluence, HEK293 cells were cotransfected with 1 μg of the pAPN-Flag plasmid (or the control vector). At 24 hpt, the cells were infected with PDCoV at an MOI of 100 at 37°C. After 2 h, we detected the internalized virions by immunofluorescence specific for the viral N protein. Each experiment was repeated at least three times. (F) The internalization ratios were analyzed as the number of cells with internalized virions/total cell numbers. Error bars represent the SD. *, P < 0.05.