Figure 1.
Sindbis virus genome-wide RNAi screen.
A. Strategy for initial and follow-up screens of Sindbis host factors. Initial screen was performed using pooled siRNAs and SINV infection at low multiplicity for 24 h. Selected siRNAs were tested individually under the same conditions; validation refers to those host factors in which ≥2/3 siRNAs inhibited or promoted SINV infection. Pooled siRNAs were also tested for effects on cell viability and for inhibition of single-cycle SINV infection. B. Ingenuity Pathway analysis of 55 proteins identified by the screen as promoting SINV-Luc infection. The top 14 overrepresented categories of molecular and cellular functions are shown, with the significance (p-values) and number of proteins indicated for each category in parentheses.
Figure 2.
Silencing of ARCN1, FUZ, and TSPAN9 in human U-2 OS cells inhibits alphavirus infection.
A. U-2 OS cells were transfected with the indicated single siRNAs. At 48 h post-transfection, cells were infected with SINV-Luc (MOI = 1) and luciferase expression was quantitated 24 h post-infection. Data were normalized to the non-targeting siRNA control (NT). Bar graph represents the mean +/− SEM of 3 determinations (*** indicates p-values<0.001). B. Quantigene assay of mRNA levels in U-2 OS cells 48 h after transfection with the indicated siRNAs, with data normalized to non-transfected control cells.
Figure 3.
Tests of ARCN1, FUZ, and TSPAN9 with other host cells and viruses.
A. HeLa cells were transfected as in (Fig. 2A), infected with SINV-GFP (MOI = 50) and infection quantitated at 24 h post-infection by fluorescence microscopy. B. U-2 OS cells were transfected as in Fig. 2A and infection by the indicated viruses was tested as detailed in the methods. C. Primary human umbilical vein endothelial cells were transfected with the indicated siRNAs, incubated for 60 h, infected with SFV or CHIKV, and infection quantitated at 8–12 h post-infection by fluorescence microscopy. Values in A–C were normalized to a non-targeting siRNA control (NT), and the data in each panel represent the mean +/− SEM of 3 independent experiments (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
ARCN1 depletion inhibits alphavirus binding to host cells.
U-2 OS cells were transfected as in Fig. 2A and tested as follows: A. Virus fusion-infection. SINV or SFV was pre-bound to cells on ice and treated at low pH to trigger virus fusion with the plasma membrane. Infected cells were quantitated by immunofluorescence. B. Cell surface binding. SINV or SFV was bound to cells on ice and detected by immunofluorescence. A–B each represent the mean +/− SEM of 3–4 independent experiments with data normalized to the NT control (*p<0.05, **p<0.01).
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).
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).