Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry
Figure 6
TSPAN9 depletion inhibits alphavirus membrane fusion.
U-2 OS cells were transfected as in Fig. 2 A and tested as follows: A. Assay of the low pH-triggered E1 conformational change. Cells with pre-bound SFV were incubated at 37°C for 20 min to permit endocytosis. Cells were fixed and permeabilized, and the low pH conformation of viral E1 was quantitated in cells by staining with mAb E1a-1. Control shows cells in which the 37°C incubation was carried out in medium containing 20 mM NH4Cl to block endosome acidification. B. Virus fusion assay. Cells with pre-bound DiD-labeled SFV were incubated at 37°C for 20 min, fixed, and the signal from fused virus particles quantitated by fluorescence microscopy. Cells were then permeabilized, stained with mAb E1a-1, and the signal from the low pH conformation of E1 quantitated. Data from the DiD lipid mixing fusion assay were corrected for the difference in the E1a-1-positive signal in the TSPAN9-depleted cells. Panels A–B 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).