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. 2021 Sep 27;95(20):e00355-21. doi: 10.1128/JVI.00355-21

FIG 7.

FIG 7

Two- and three-component replicases of SINV and CHIKV containing inactive protease are capable of RNA synthesis. (A) Schematic presentation of the replicases used. C1 shows the P1234 expressed from a single-component CMV-P1234 plasmid, C2 shows the P123 and nsP4 expressed by the two-component replicase, and C3 shows nsP1, P2CA3 and nsP4 expressed by the three-component replicase. (B). HEK293T cells in 96-well plates were cotransfected with CMV-P12CA3-CHIKV, CMV-ubi-nsP4-CHIKV, and HSPolI-FG-CHIKV or CMV-P12CA3-SINV, CMV-ubi-nsP4-SINV, and HSPolI-FG-SINV plasmids. As the negative controls, CMV-P1234GAA, which lack polymerase activity, were used. The amounts of CMV-P12CA3 and HSPolI-FG plasmids were kept constant, while the CMV-ubi-nsP4 plasmid was provided in a 1:10, 2:10, 4:10, 6:10 8:10, 1:1, 2:1, 4:1, 6:1, or 8:1 molar ratio in respect to CMV-P12CA3. Cells were incubated and the data were collected and analyzed as described for Fig. 2B. Boosts of Fluc (replication, left panel) and Gluc (transcription, right panel) activities are shown as the means ± SD of three independent experiments. (C). HEK293T cells in 96-well plates (left) or in 24-well plates (right) were cotransfected with matching combinations of CMV-P12CA3-CHIKV, CMV-ubi-nsP4-CHIKV, and HSPolI-FZsG-CHIKV. As the negative control, CMV-P1234GAA-CHIKV was used instead of CMV-P12CA3+CMV-ubi-nsP4. This experiment was performed and the data were collected, analyzed, and presented as described for Fig. 3B. (D) U2OS cells in 12-well plates were cotransfected with single-, two-, and three-component replicases as indicated on the x axes. For two- and three-component replicases, all plasmids encoding ns-proteins were combined in equimolar amounts. As negative controls, CMV-P1234GAA were used instead of CMV-P1234. Cells were incubated at 37°C and lysed for 18 h p.t. Fluc (replication, left panel) and Gluc (transcription, right panel) activities produced by active replicases were normalized to the P1234GAA controls. The values obtained for the P1234GAA controls were taken as 1. The means ± SD of three independent experiments are shown; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (Student’s unpaired t test).