FIG. 8.

Analysis of effects of mutations in the VEEV 5′UTR on the translation of the encoded proteins and RNA replication. (A) Schematic representation of the firefly luciferase-encoding constructs used for evaluating the effect of the 5′UTR-specific mutations on template translation. A detailed description of the constructs is presented in Materials and Methods. Shown are values for firefly luciferase expression in BHK-21 cells transfected with the in vitro-synthesized, capped RNAs. One microgram of each template was mixed with 0.1 μg of Renilla luciferase-encoding RNA and electroporated into BHK-21 cells as described in Materials and Methods. Equal aliquots of transfected cells were seeded into 35-mm dishes, and luciferase activities were determined at the indicated time points using a dual-luciferase system (Promega). (B) Schematic representation of DI RNAs having mutations in the 5′UTR and helper VEEV replicon used in the study. BHK-21 cells were cotransfected with 2 μg of replicons and 2 μg of the indicated DI RNAs. Equal numbers of electroporated cells were seeded into 35-mm dishes and incubated at 37°C in 5% CO₂. At the indicated time points, cells were lysed, and luciferase activity was measured as described in Materials and Methods. PEP, postelectroporation; RLU, relative light units.