FIG. 2.

Extensive modifications of the loop sequence do not affect virus replication. (A) Computer-predicted secondary structures of the 5′ termini in the genomes of VEE/SINV variants used in the experiments, infectivities of the in vitro-synthesized RNAs in the infectious center assay, and virus titers at 24 h post-RNA transfection. Open circles indicate positions of the mutations. PEP, postelectroporation. (B) Autoradiograph of the dried gels with the RNAs metabolically labeled with [³H]uridine. Equal numbers of BHK-21 cells were infected with the indicated VEEV variants at an MOI of 10 PFU/cell. The RNAs were metabolically labeled with [³H]uridine (20 μCi/ml) in the presence of 1 μg of dactinomycin/ml between 4 and 8 h postinfection and then isolated and analyzed by agarose gel electrophoresis under denaturing conditions as described in Materials and Methods. Bands corresponding to genomic and subgenomic RNAs were visualized by autoradiography. G and SG indicate positions of the viral genomic and subgenomic RNAs, respectively. RNA bands were excised, radioactivity in the genomic and subgenomic RNAs was measured by liquid scintillation counting, and the molar SG-to-G ratios are presented. (C) BHK-21 cells were infected with the indicated variants at an MOI of 10 PFU/cell. Virus replication was assessed as described in Materials and Methods.