FIG. 4.

MNV-1 infection is clathrin independent. (A) RAW 264.7 cells were infected with NR-containing virus at an MOI of 0.001 and rocked at room temperature for 60 min. RAW 264.7 cells were either pretreated (pretreatment) or treated for 60 min pi (posttreatment) with 40 μM chlorpromazine (Chloro) or 300 mM sucrose. (B) RAW 264.7 cells were transfected with a GFP-only (GFP), GFP-tagged wt (EPS 15 wt), or DN construct (EPS 15 DN) of EPS 15 and then infected with MNV-1 at an MOI of 10 for 12 h. The number of VPg-expressing cells was determined by immunofluorescence and normalized to the value for the GFP-only control. (C) RAW 264.7 cells were incubated with an Accell siRNA clathrin heavy-chain construct (CHC siRNA) or a nontargeting construct (NT siRNA) (Dharmacon) for 72 h. Cells were then infected with MNV-1 or VSV at an MOI of 10. The number of VPg-expressing cells was determined by immunofluorescence 12 h after infection and normalized to the value for the NT control. (D) To verify clathrin heavy-chain protein knockdown, protein samples from cells expressing each siRNA construct were analyzed by immunoblotting for clathrin heavy chain, and protein levels were quantitated as described in the text. *, P < 0.05; ***, P < 0.001.