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. 2020 Aug 16;12(8):896. doi: 10.3390/v12080896

Figure 6.

Figure 6

Establishment of a minigenome system for LCMV strain WE (LCMV-WE): (a) a schematic diagram of SMG-luc or -GFP and its replication, transcription, and translation processes. A cDNA fragment containing the S 5′ UTR, S IGR, renilla luciferase, or GFP ORFs in an antisense orientation with respect to the 5′ UTR and the S 3′ UTR were cloned between the murine pol I promoter and the terminator of a pRF vector. An additional G residue was inserted between the murine pol I promoter and the viral genome sequence. The SMG plasmids are transcribed by the polymerase I RNA polymerase to generate cRNA in BHK cells. The cRNAs are then encapsidated with nucleoprotein (NP). vRNAs are produced in the presence of L protein (L). The encapsidated cRNA is transcribed into reporter gene mRNA. Finally, transcribed mRNA is translated by host-cell machinery to produce reporter proteins (luciferase or GFP). (b) BHK-21 cells were transfected with minigenome plasmids SMG-luc, either pC-NP or pCAGGS, either pC-L or pCAGGS, and pC-Fluc. The transfected cells were incubated for 2 days at 37 °C, and then renilla luciferase activity was measured using the Renilla Luciferase Assay System and firefly luciferase activity was measured using the Bright-Glo Luciferase Assay System. (**** p < 0.0001) Error bars indicate standard deviations. The experiments were performed three times independently.