An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication
Figure 5
Efficiency of translation initiation at L uAUG is determined by its sequence context.
A. L 5′-UTR GFP mRNA reporter constructs were generated such that the uORF is fused in frame to GFP. The nucleotide sequence immediately surrounding the uAUG of each construct is displayed. The first construct (uORF-GFP) includes the first 46 nucleotides of the L 5′-UTR up to the uAUG followed by the entire L uORF sequence placed in frame with the GFP ORF (labeled uORF-GFP). The middle construct (uORF-GFP SK) is identical to the first, but the uAUG is surrounded by a strong Kozak sequence (A at the −3 position and a G at the +4 position, where the A of the AUG is designated as +1). The bottom construct includes the L 5′-UTR, through the uAUG and the second codon of the uORF which was placed in frame with the GFP ORF, but lacks the rest of the uORF. In each case, the start codon for GFP was removed. The number of nucleotides in each construct is indicated and the features of each construct are summarized in the box above the diagram. B. Equal amounts of each in vitro transcribed mRNA were transfected into 293T cells. At 2.5 hours post transfection, cells were harvested and analyzed by flow cytometry. The experiment was performed in triplicate, and a representative sample is displayed for each group. C. GFP mRNA levels, as determined by real time PCR, present in the transfected cells described in B were determined for each sample.