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. 2020 Sep 4;16(9):e1008825. doi: 10.1371/journal.ppat.1008825

Fig 3. Alphavirus replicases can replicate and transcribe their templates in Aedes albopictus cells.

Fig 3

(A) C6/36 cells in 12-well plates were co-transfected with matching pairs of Ubi-P1234 and AlbPolI-FG plasmids. At 12, 24. 36, 48, 54, 66, 72 and 144 h p.t. growth media aliquots were collected the activity of secreted Gluc measured. Means of relative luminescence units (RLU) per 1 μl of sample + SD of three independent experiments are shown. (B) C6/36 cells in 96-well plates were co-transfected with matching pairs of AlbPolI-FG and either Ubi-P1234 or Ubi-P1234GAA plasmids. Cells were incubated at 28°C and lysed 48 h p.t. The Fluc (left panel) and Gluc (right panel) activities produced by P1234 replicases were normalized to the polymerase-defective P1234GAA controls as described for Fig 2. Means + SD of three independent experiments are shown. (C) C6/36 cells in 12-well plates were co-transfected as described for panel A. Total RNA was extracted and positive strand RNAs were detected using northern blotting. “Genomic” indicates the full-length template RNA. Note that transcripts made by mosquito RNA polymerase I using AlbPolI-FG plasmids as template co-migrate with replicase-generated positive-strand genomic RNAs. “Subgenomic” indicates the SG RNAs synthesized by replicases using the SG promoter. The experiment was repeated twice with similar results; data from one experiment is shown. (D) HEK293T cells (left panel) were co-transfected with HSPolI-FZsG-CHIKV and CMV-P1234-CHIKV; C6/36 cells (right panel) were co-transfected with AlbPolI-FZsG-CHIKV and Ubi-P1234-CHIKV. Control cells were transfected with plasmid expressing EGFP from a CMV promoter (HEK293T) or from a polyubiquitin promoter (C6/36). At 18 h (HEK293T) or 48 h (C6/36) p.t. cells were collected and analyzed with an Attune NxT Acoustic Focusing Cytometer. The number of ZsGreen-expressing cells is shown as a proportion of the number of EGFP expressing cells, to control for the different transfection efficiency of the two cell types. All transfections were performed in triplicate, means + SD are shown.