Figure 7.

Evaluation of the efficiency of viral genome transcription, replication, and packaging in virus-like particles (VLPs): (a) Viral genome transcription and replication were assessed in BHK-21 cells transfected with SMG-luc, SMG-5UTRΔ20–40-luc, SMG-5UTRΔ41–60-luc, SMG-5UTRΔ60–77-luc, SMG-3UTRΔ20–38-luc, SMG-3UTRΔ39–60-luc, or SMG-UTR-comple-luc under conditions of the co-expression of NP, L protein, GPC, and Z protein. BHK-21 cells were transfected with each mutated SMG-luc, pC-NP, pC-L, pC-Z, and pC-GPC. As a background control for the rescue of LCMV RNA analogs into VLPs, cells transfected with SMG-luc, pC-NP, and pC-L were used (SMG-luc (no VLPs)). After incubation for 48 h at 37 °C with 5% CO2, luciferase expression was examined. (b) The expressions of LCMV NP and α tublin in transfected cells were confirmed by Western blot analysis. (c) Packaging of viral genome RNA analogs derived from SMG-luc, SMG-5UTRΔ20–40-luc, SMG-5UTRΔ41–60-luc, SMG-5UTRΔ60–77-luc, SMG-3UTRΔ39–60-luc, or SMG-UTR-comple-luc into VLPs were assessed. Supernatants from each well of Figure 7a were harvested and used to infect fresh monolayers of BHK-21 cells, which were then incubated for 4 h at 37 °C before adding helper LCMV. Ninety hours postinfection, the passage culture was examined for luciferase expression. The packaging efficiency of each SMG was determined by dividing the relative light units (RLUs) value obtained from the passage culture 90 h postinfection by the RLUs value obtained in Figure 7a. (** p < 0.01, and **** p < 0.0001). Error bars indicate standard deviations. All experiments shown were conducted three times independently.