ESCRT regulators control CCHFV entry.

<p>(A) To generate VSV pseudotyped with CCHFV G (VSV-CCHFVG), 293FT cells were transfected with either pBabe-βGal (control) or pC-G. After 18 h, the cells were inoculated with a VSV-VEEVGP stock for 6 hours. The supernatants were collected 48 h after infection and incubated with SW13 cells to determine the titer of the pseudotyped virus. Luciferase activity was measured 24 h later. (B) A neutralization assay was performed by incubating VSV-CCHFVG or VSV-VEEVGP with anti-Gc antibody at the indicated dilutions for 30 min. The pseudotype-antibody mixtures were subsequently added to SW13 cells, and luciferase activity was measured 24 h later. (C) SW13 cells were preincubated with DMSO or bafilomycin A (20 nM) for 1 h and then challenged with VSV-CCHFVG in the presence of the drug. Luciferase activity was measured after 24 h. (D) SW13 cells were treated with siRNAs as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004390#ppat-1004390-g004" target="_blank">Fig. 4</a>. Forty-eight h later, cells were plated for assessment of host gene silencing or pseudotype infection. The following day, cells were incubated with VSV-CCHFVG or were tested for host gene silencing by immunoblotting (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004390#ppat-1004390-g004" target="_blank">Figure 4C</a>). Luciferase activity was measured 24 h after pseudotype addition to cells. The averages of three experiments and standard deviations are reported relative to mock.