FIG 2.

Single- and two-component replicases of alphaviruses have similar activities. (A). Schematic presentation of expressed replicase precursors. C1, single-component replicase from plasmids CMV-P1234-SINV and so on; C2, two-component replicase from plasmids CMV-P123-SINV+ CMV-ubi-nsP4-SINV and so on. (B) HEK293T cells in 96-well plates were cotransfected with matching pairs of CMV-P1234 and HSPolI-FG plasmids (C1) or with matching combinations of CMV-P123, CMV-ubi-nsP4 (in a 1:1 molar ratio), and HSPolI-FG plasmids (C2). As the negative control, CMV-P1234^GAA, which encodes polyprotein lacking RNA polymerase activity, was used instead of CMV-P1234. Cells were incubated at 37°C and lysed for 18 h p.t.; cells transfected with plasmids containing sequences from EILV were incubated at 28°C and lysed for 48 h p.t. Fluc (marker of replication) and Gluc (marker of transcription) activities produced by active replicases were normalized to the P1234^GAA controls. The value obtained for the P1234^GAA controls was taken as 1. The means ± standard deviation (SD) of three independent experiments are shown. (C) HEK293T cells in 12-well plates were cotransfected with matching combinations of CMV-P123, CMV-ubi-nsP4 (in 1:1 molar ratio), and HSPolI-FG plasmids; control cells were mock-transfected. Cells were incubated as described for panel B, after which total RNA was extracted and analyzed 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 comigrate 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.