FIG. 6.

Replication of VEEVubiΔ3+4 variants, containing combinations of mutations identified in efficiently replicating pseudorevertants, in BHK-21 and C₇10 cells. (A) Schematic representation of VEEVubiΔ3+4 genomes with different mutations and infectivities of the in vitro-synthesized viral RNAs in the infectious center assay. Solid boxes indicate nsP1 sequence containing clustered silent mutations. (B) Single-step viral growth curves after electroporation of 3 μg of in vitro-synthesized RNAs into BHK-21 cells. At the indicated times, the medium was replaced and virus titers were determined in BHK-21 cells, as described in Materials and Methods. The dashed line indicates the detection limit. (C) Synthesis of virus-specific RNAs in the transfected BHK-21 cells. At 2 h post transfection, viral RNAs were metabolically labeled with [³H]uridine for 6h, as described in Materials and Methods, and analyzed by agarose gel electrophoresis. Positions of the genomic and subgenomic RNAs are indicated.