FIG. 2.

The ORF7b protein is translated via ribosome leaky scanning. (A). Schematic diagram of the cDNA constructs used to assess the translation of ORF7b. Gene 7 constructs included wild-type sequence (7ab), an optimal translation initiation consensus sequence at the ORF7a initiation codon (7ab 7a Kozak), a point mutation within the ORF7a coding region creating an out-of-frame ATG that does not alter the ORF7a amino acid sequence (7ab ATG), two point mutations within the ORF7a ORF resulting in two in-frame stop codons (7ab 7a Stop), a point mutation eliminating the ORF7a stop codon (7ab 7a Stop KO), a point mutation eliminating the ORF7a initiation codon (7ab 7a Start KO), and a point mutation eliminating the ORF7b initiation codon (7ab 7b Start KO). All ATG sequences are boxed, and stop codons are italicized. (B) 293T cells were transfected with plasmids encoding the indicated cDNAs and analyzed for ORF7a (horizontal axis) and ORF7b (vertical axis) expression by flow cytometry at 18 h posttransfection. (C) 293T cells were transfected with plasmids encoding the indicated cDNAs, lysed 18 h posttransfection, and analyzed for ORF7b and β-actin expression by Western blotting. (D) The ORF7b protein band intensities from panel C were quantified and normalized to β-actin expression by using phosphorimager analysis. The quantified data are from five independent experiments.