Fig. 6

EXi-RVFV Depletion Eliminates Interferon-B Induction in Naïve Recipient Cells. A The sucrose density fractions containing EXi-RVFV, the most bottom fraction containing virus (density of 1.25 g ml⁻¹), and the working stock of MP12 strain (1.97 × 10⁷ pfu/ml), were analyzed by western blot for the exosome marker CD9. B EXi-RVFV samples were subjected to immunoprecipitation using magnetic Dynabeads coated with human anti-CD9 antibody. The efficacy of pull-down was analyzed by anti-CD9 immunoblot of the starting material (Input EXi-RVFV), the immunoprecipitated EXi-RVFV, and the EXi-depleted supernatant that was left behind (ΔEXi). Three biological repeats were performed. A typical western result is shown. C Naïve U937 cells were either left untreated, or were subjected to one of the following treatments: i) Poly(I:C); ii) EXu; iii) EXi-RVFV; iv) ΔEXi. The IFN-B expression levels in treated cells were then quantified by RT-qPCR analysis. For each treatment condition, mean value ± SEM from three biological replicates is shown (***P ≤ 0.005). D Anti-IFN-B immunoblot analysis was performed to compare IFN-B induction in naïve U937 cells treated with either EXi-RVFV or samples depleted of EXi-RVFV by anti-CD9 IP pulldown (ΔEXi). The intracellular IFN-B expression was analyzed at 24 h post treatment. Untreated cells, cells treated with EXu, or cells infected with either the MP12 strain or the ΔNSs mutant strain of RVFV, were also analyzed side by side. For each treatment condition, mean values ± SEM from three biological replicates are shown in the density bar graph at the bottom. ****P ≤ 0.0005. E Western blot analyses of the viral G protein and the viral N protein were performed for immunoprecipitated EXi-RVFV obtained by CD9 pulldown and the resulting supernatant depleted of exosomes (ΔEXi)