Figure 4.

Analysis of trans-activating frameshift stimulation by nsp1β mutants. Plasmids expressing SD01-08 PRRSV WT or mutant nsp1β were co-transfected with a plasmid expressing nsp2 in RK13 cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. As controls, single expression of nsp2 or nsp1β, expression of a self-cleaving nsp1β-2 polyprotein, and a nontransfected sample were included. A, following ³⁵S metabolic labeling, proteins were immunoprecipitated with mAb58-46 (nsp2, nsp2TF, and nsp2N) or mAb-FLAG (nsp1β) and analyzed by SDS-PAGE and autoradiography. Size markers and the positions of bands for nsp2, nsp2TF, nsp2N, and nsp1β are indicated beside each panel. B, band intensities were quantified by phosphor imaging and corrected for amino acid content and Met/Cys incorporation efficiency, after which the nsp2, nsp2TF, and nsp2N levels were used to calculate ribosomal frameshifting efficiencies.