FIG. 4.

Analyses following the transfection of RNA derived from pOR with the S codon at position 1555 of the ORF exchanged for an F codon. (A) Detection of cells containing replicating virus resulting from RNA transfection. Cells were transfected with equal doses of the in vitro-transcribed RNAs by the DEAE-dextran method and seeded at a sufficient density that 3 days posttransfection a closed layer of cells was obtained. At this time point, cells were fixed and virus-containing cells were visualized by MAb-mediated peroxidase staining. Below the dishes, the NS2-3 cleavage efficiency determined for the respective polyprotein after transient expression is indicated. (B) Analysis of part of the NS2 sequence from RNA obtained at different time points after transfection of RNA derived from pOR/S-F. Cells were split every 3 to 4 days after transfection, and total RNA was prepared from each passage and used as starting material for RT-PCR. RNA from passage 0 was isolated 3 days posttransfection. The amplified fragments were directly sequenced to look for the mutation introduced into pOR/S-F (arrowhead). In lanes C, the sequence of the RT-PCR product derived from the in vitro-transcribed RNA is shown as a control.