FIG. 7.

Construction and characterization of replicon 5′-CS-A/ORF. (A) Schematic drawing of the construction of 5′-CS-A/ORF from replicon C17 (not to scale). 5′-CS-A was deleted from its original position and used to replace the 5′-CS-B element within the ORF, which was not frame shifted by this manipulation. (B) Immunofluorescence analysis of cells transfected with RNA of replicon C17 (positive control), 5′-CS-A/ORF, or ΔNS5 (negative control); Immunofluorescence staining was performed 3 days posttransfection with a monoclonal antibody recognizing protein NS1. (C) Replication kinetics of 5′-CS-A/ORF and control RNAs. RNA isolated from 2,000 cells was quantified at various time points posttransfection by real-time PCR. The logarithmic means from two experiments are shown (error bars indicate maximum-minimum values). (D) Average increase or decrease of RNA copies in percent per hour between the individual measurement points of the experiment.