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. 2018 Jun 5;10(6):306. doi: 10.3390/v10060306

Figure 2.

Figure 2

Figure 2

Effect of NH125 on virus-driven luciferase expression and infectious VSV titers. (a) Vero and BHK-21 cells were treated for 1 h with NH125 using the indicated concentrations, washed with medium, and subsequently inoculated for 1 h with VSV*∆G(sNLuc) using an m.o.i. of 0.5 ffu/cell. Virus which had not entered the cells during the incubation period was neutralized with a monoclonal antibody directed to the VSV G protein. Secreted NanoLuc luciferase activity was determined in the cell culture supernatant 6 h p.i. The percentage RLUs relative to DMSO-treated VSV*∆G(sNLuc)-infected cells were calculated. Mean values and standard deviations of six parallel experiments are shown. Asterisks indicate significant inhibition of virus entry (compared to DMSO-treated cells). (b) Vero and BHK-21 cells were inoculated for 6 h with VSV*∆G(sNLuc) (m.o.i. of 0.5 ffu/cell) and washed to remove sNLuc reporter protein which has been secreted up to this time point. Subsequently, the cells were incubated for 5 h with NH125 at the indicated concentrations. Luciferase activity was determined as above. (c) BHK-21 cells treated for 2 h with either DMSO (0.1%, v/v) or NH125 using the indicated concentrations and subsequently infected for 1 h with VSV* (m.o.i. of 0.001 ffu/mL) and subsequently maintained for 24 h in the presence of NH125 or DMSO. GFP fluorescence indicating infected cells was detected by fluorescence microscopy. (d) Infectious VSV* titers released into the cell culture supernatant of infected BHK-21 and Vero cells following treatment with the indicated concentrations of NH125. Mean titers and standard deviations of three infection experiments are shown and expressed as fluorescent focus-forming units per ml (ffu/mL). (e) VSV* was treated at 37 °C for 2 h with either DMSO or NH125 (10 µM) prior to ultracentrifugation. Infectious virus titers were subsequently determined on BHK-21 cells. Mean titers and standard deviations of three independent experiments are shown. (f) VSV* was passaged on Vero cells in the presence of NH125 (10 µM). The infectivity of passage 5 virus compared to non-passaged VSV was determined on BHK-21 cells that have been treated with NH-125 (10 µM).