The presence of an adenine residue at position −1 of the SG promoter of SFV increases template transcription. (A) Organization of the plasmids used in the trans-replication assay. The substitution of the −1 guanine residue in the SG promoter with adenine, which yielded the HSPolI-FG-SFV-SG−1G/A template-expression plasmid, is shown in the drawing. The other designations are the same as in Fig. 6A. (B) U2OS cells in 12-well plates were cotransfected with 500 ng of HSPolI-FG-SFV or HSPolI-FG-SFV-SG−1G/A and with 500 ng CMV-P1234-SFV (WT) or with a plasmid carrying the swaps and point mutations indicated on the horizontal axis. As a negative control, CMV-P1234GAA-SFV, which lacks polymerase activity, was used. The cells were incubated at 37°C, and aliquots of the culture supernatants were collected at 16, 20, and 24 h p.t. The activities of Gluc produced in the presence of the analyzed replicases were normalized to those of the P1234GAA controls. The means ± SD of three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (Student’s unpaired t test).