Fig 2. Alphavirus replicases can replicate and transcribe their templates in human cells.

(A) HEK293T cells in 12-well plates were co-transfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids. At 4, 8, 12, 16, 20, 22, 24 and 48 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 + standard deviation (SD) of three independent experiments are shown. (B) HEK293T cells in 96-well plates were co-transfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids and, as negative control, CMV-P1234^GAA, which lacks polymerase activity, instead of CMV-P1234. Cells were incubated at 37°C and lysed 18 h post transfection (p.t.); cells transfected with plasmids containing sequences from EILV were also incubated at 28°C and lysed 48 h p.t. Fluc (marker of replication, left panel) and Gluc (marker of transcription, right panel) activities produced by active replicases were normalized to the P1234^GAA controls. Value obtained for P1234^GAA controls was taken as 1; activities lower than that observed for P1234^GAA are also shown as 1. Means + SD of three independent experiments are shown; p<0.01**; p<0.001*** (Student's unpaired t-test). (C) HEK293T cells in 12-well plates were co-transfected and incubated as described for panel A; control cells were mock-transfected. Total RNA was extracted and analysed by northern blotting. Full-length “genomic” template RNA of positive (+) and negative (-) polarity and subgenomic RNA are indicated. Note that transcripts made by human RNA polymerase I using HSPolI-FG plasmids as templates co-migrate with replicase-generated positive-strand genomic RNA and are detected by the same probe. The experiment was repeated twice with similar results; data from one experiment is shown.