FIG 5.

Analysis of frameshift stimulators. (A) Frameshifting efficiencies measured using dual-luciferase constructs. BHK-21 cells were transfected with frameshift reporter constructs and 18 h later were either infected with WT virus at an MOI of 10 or mock infected. Lysates were harvested at 7 h p.i. and assayed for Renilla and firefly luciferase activity. Frameshift efficiencies were determined by comparing luciferase activities to an in-frame control (IFC) construct. Mean values and standard deviations are shown, each based on nine separate transfections. (B) Western blot verifying infection of infected samples. Aliquots of each of the cell lysates were separated on a 10 to 20% Tris-Tricine gradient gel and probed with rat monoclonal anti-tubulin (red, IRDye 700-labeled secondary) and mouse monoclonal anti-VP1 (TMEV capsid protein) (green, IRDye 800-labeled secondary) antibodies. Note that the rat monoclonal primary cross-reacts with both the secondary antibodies. (C) Schematic representation of the fragments cloned into the pIDluc vector. All constructs contain the U-to-C mutation removing the −1 frame UAA stop codon (red) to allow expression of the downstream luciferase and the C-to-U mutation to introduce a zero-frame UAA stop codon just 3′ of the shift site (see the text). pIDluc IFC contains an extra U in the G_GUU_UUU shift site sequence (blue). (D) Frameshifting efficiencies of dual-luciferase constructs containing stem-loop mutants. See the description of panel A for details. Mean values and standard deviations are shown, each based on nine separate transfections.