Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry
Figure 5
FUZ depletion inhibits alphavirus endocytosis.
U-2 OS cells were transfected as in Fig. 2 A and tested as follows: A. SFV endocytosis. Cells with pre-bound SFV were incubated at 37°C for 30 min to permit endocytosis, and then fixed and stained with rabbit antibody to the envelope proteins before and after cell permeabilization, conditions that detect virus remaining on the cell surface (red/yellow) vs. internalized virus (green), respectively. B. Transferrin uptake. Cells were pre-bound with fluorescent transferrin on ice, incubated for 30 min at 37°C to permit endocytosis, and washed at pH 2.5 to strip off non-internalized transferrin before fixation. The control was acid-washed prior to 37°C incubation. C. Fluid phase uptake. Cells were imaged after incubation with fluorescent dextran for 3 h at 37°C followed by a 2 h chase. Panels A–C show confocal extended focus images (bar = 10 µM). Bar graphs with each panel represent the mean +/− SEM of 3 independent experiments with data normalized to NT control (*p<0.05, **p<0.01, ***p<0.001). All three siRNAs to FUZ and TSPAN9 produced similar results (data not shown).