Figure 6: Cholesterol depletion of producer cells decreases infectivity of budding viruses.

Vero cells infected for 24 h with JUNV were mock treated (a) or treated with 10 mM Methyl-β-Cyclodextrin (MβCD-treated; b) for 1 h at 37 °C. Cells were then extensively washed with PBS and incubated for an additional 12 h at 37 °C. Subsequently, the supernatant was harvested and stained with LD05 GPC antibody conjugated to Alexa Fluor 647 and CD9 antibody conjugated to PE as in Fig. 5a. Dot plots show CD9 antibody fluorescence as a function of Alexa 647 GPC antibody staining. Percentages in each corner indicate the proportion of each population in the given quadrant. The data shows that MβCD treatment strongly decreases CD9 at the surface of virions. (c) JUNV-A647 particles from MβCD or mock-treated producer cells were sorted and 8,000 particles per conditions were used to infect Vero cells for 16 h. Subsequently, the Vero cells were stained with a viral NP-specific antibody conjugated to an Alexa Fluor 647 and analysed by flow cytometry to measure the percentage of infected cells. A twofold decrease in infectivity was observed when producer cells were treated with MβCD compared to the mock condition. Error bars are the mean±s.d. of duplicates where at least 10,000 cells per sample were acquired. The difference marked by the star is significant (unpaired t-test P value=0.024).