FIG 5 .

The presence of fusogenic spike protein at the plasma membrane is required to induce macropinocytosis. (A) Cells were mock infected or infected with MHV A59, 2S, or C12 at an MOI of 1 PFU/cell. Nanoparticles were added at 5 hpi for 3 h, and the cells were washed, fixed, stained, and imaged. (B to D) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell in DMEM or in DMEM with DMSO or dec-RVKR-cmk (dRc) for 8 h. The cells were exposed to dec-RVKR-cmk for 12 h, and toxicity was assessed with CellTiter-Glo (B). Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Percentages of syncytial cells (C) and cells with internalized nanoparticles (D) were measured. (E) Cells were mock infected or infected with MHV A59 or 2S at an MOI of 1 PFU/cell. At 5 hpi, cells were treated with TPCK trypsin for 5 min and washed and then nanoparticles were added for 3 h. Cells were washed, fixed, stained, and imaged. Data are represented as the mean ± the standard error of the mean of two replicates performed in duplicate. n = ≥30 cells per replicate. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test; ***, P < 0.0001; **, P < 0.01; *, P < 0.05.