Figure 3.

Effect of DDD85646 on different steps of the LCMV life cycle. (A) Time-of-addition assay: Vero E6 cells were seeded into a 96-well plate at a density of 2 × 10⁴/well. The next day, the cells were infected with the single-cycle infectious rLCMV∆GPC/ZsG (MOI 0.5) and treated with DDD85646 (5 µM) or with VC, starting 1 h before (−1 h) or 2 h after (+2 h) infection. The LCMV cell-entry inhibitor F3406 (10 µM) and ribavirin (Rib) (100 µM) were used as controls. At 48 h pi, ZsG⁺ cells were assessed using the Synergy™ H4 Hybrid Microplate Reader from Biotek. Values were normalized to VC-treated infected cells; the data represent an average of three biological replicates. (B) LCMV cell-based MG assay: HEK293T cells were seeded into M24-well plates and transfected with plasmids expressing LCMV MG-CAT together with plasmids expressing the viral trans-acting factors NP, L polymerase, and T7 RNA polymerase and then treated with DDD85646 (5 or 10 µM); Rib- and VC-treated samples were used as controls. At 72 h post-transfection, cell lysates were prepared, and total protein was determined using a BCA protein assay. The same amount of total protein from each sample was used to determine CAT protein expression levels using a CAT ELISA kit (Roche, Sydney, Australia) and normalized by assigning the value of 100% activity to vehicle control-treated samples. (C) Z budding activity assay: HEK293T cells were seeded onto poly-L-lysine-coated M12-well plates at 1.75 × 10⁵ cells/ well. The next day, the cells were transfected with either pC-LCMV-Z-GLuc, pC-LASV-Z-Gluc, or pC-LCMV-Z-G2A-GLuc. At 5 h post-transfection, the cells were washed three times and fed with fresh medium containing the relevant drugs at the indicated concentrations. At 48 h post-transfection, cell culture supernatant (CCS) samples were collected, and whole-cell lysates (WCL) were prepared. GLuc activity in the CCS and WCL samples was determined using the SteadyGlo Luciferase Pierce: Gaussia Luciferase Glow assay kit and a Berthold Centro LB 960 luminometer (Berthold Technologies, Oak Ridge, TN, USA). The activity (relative light units) of GLuc in the CCS and WCL was used as a surrogate for Z expression and budding efficiency, defined as the ratio Z_VLP/Z_VLP + Z_WCL. Budding efficiency values were normalized by assigning the value of 100% to vehicle control-treated samples and plotted using Prism10. (D) Virucidal assay: 10⁵ FFU of rLCMV/GFP-P2A-NP was incubated for 30 min at RT in the presence of 0, 5, or 10 µM DDD85646 or in the presence of the validated virucidal compound sodium hypochlorite (0.5%). After treatment, the samples were diluted 1000-fold in DMEM/2%FBS, resulting in concentrations (5 nM and 10 nM) that did not have noticeable anti-LCMV activity, and the number of infectious particles was determined by the FFA.