Figure 2.

Effect of the NMT inhibitor DDD85646 on LCMV multi-step growth kinetics and peak titers in A549 cells. (A) Effect of DDD85646 on the production of infectious viral progeny. A549 cells were seeded at 2 × 10⁵ cells/well in an M12-well plate, infected with rLCMV/GFP-P2A-NP (MOI 0.05), and treated with DDD85646 (5 or 10 µM) or with VC. At the indicated time points, cell culture supernatants were collected, and the titers of infectious virus were determined by the focus-forming assay (FFA) using Vero E6 cells. (B,C). Effect of DDD85646 on virus propagation. At the indicated h pi, samples from A were switched to FluoroBrite DMEM containing Hoechst dye (5 µg/mL) and stained for 15 min for live imaging fluorescence (B). GFP signals were determined using Cytation 5 and normalized to the average of the VC (72 h pi). (C,D) Effect of DDD85646 on viral RNA synthesis. Total cellular RNA was isolated from the samples described in (B), and the same RNA amount (10 ng) of each sample was analyzed by RT-qPCR using random hexamers for the RT step, followed by quantitative PCR with specific primers for LCMV NP and the host cell GAPDH. The repeated-measures analysis of variance with mixed-effect analysis and Dunnet’s correction for multiple comparisons were used to determine the statistical significance of the technical triplicates. Statistically significant values: **** p < 0.0001. (E) Effect of DDD85646 on LCMV replication and transcription. Cells were infected with rLCMV/GFP-P2A-NP (MOI 0.05), and at the indicated h pi, total cellular RNA was isolated and analyzed by Northern blotting using an LCMV NP-specific dsDNA ³²P probe. Methylene blue (MB) staining was used to confirm similar transfer efficiencies for all the RNA samples.