Fig. 4. Generation of DVG-A-carrying virus-like particles.

a Assessment of DVG-A packaging ability by WT virus. Infected or uninfected Vero cells were transfected with WT replicon, inactive replicon, DVG-A reporter, or DVG-A out-of-frame reporter RNA. 48 h post-transfection, the cell supernatant was collected from donor cells, depleted of naked RNA, and used to infect naïve recipient cells, which were harvested 24 h later for measuring reporter activity. Luciferase activity in recipient cells is shown. ****p ≤ 0.0001 (by two-way ANOVA with Dunnet’s multiple comparison, as compared to uninfected cells). b Schematic illustration of VLP assays: HEK-293T cells were transfected with a CPrME, E₃₀-NS1 and DVG -encoding plasmids. 72 h post-transfection the cell culture supernatant with DVG-containing VLPs was harvested. c VLP assays using WT replicon and DVG-A reporter. Donor HEK-293T cells were transfected with WT replicon or DVG-A reporter plasmid, with or without (mock) CPrME and E₃₀-NS1. The cell culture supernatant was treated with nuclease and used to infect naïve recipient Vero cells. Reporter activity in recipient cells is shown. ****p ≤ 0.0001 (by two-way ANOVA with Dunnet’s multiple comparison, as compared to mock conditions). d Quantification of DVG-A genomes in VLPs generated using native DVG-A. Clarified and nuclease-treated supernatant from donor cells were subjected to DVG-A quantification using RT-qPCR. Negative control refers to supernatant derived from cells transfected with the same amount of DVG-A-containing plasmid in the absence of CPrME or E₃₀-NS1. ***p = 0.0004 (by two-tailed t-test). All graphs show the mean and SD; n = 3 per group of a representative experiment out of three. Source data are provided as a Source Data file.