FIG 1.

(A) Schematic representation of plasmid p5′wt/3′wt_1. This plasmid allows the synthesis of mRNA 5′wt/3′wt_1, which mimics the TCRV NP mRNA. The construct, comprising the TCRV S antigenome 5′ noncoding sequence (5′NC), fused to the FLUC ORF followed by the TCRV S antigenome intergenic region (IGR), is located downstream of the T7 RNA polymerase promoter (pT7). Nonviral nucleotides preceding the 5′NC are indicated by a black triangle. The position of the SmaI (S) restriction site used for plasmid linearization is shown. (B) Alignment of the viral 5′ UTR sequence to the corresponding region of mutant mRNAs. The wild-type sequence is indicated with capital letters (5′wt/); 5′ nonviral nucleotides are marked with a black bar on top. The AUG codon is boxed; the neighboring sequence is underlined. Substitutions are indicated with lowercase letters. Unmodified viral residues are marked with asterisks. (C) Translation activity of the synthetic virus-like mRNAs. (Left) Schematic representation of synthetic transcripts. The 5′ cap structure is represented with a black circle; the H_L sequence and the position of the BsaAI sequence (B) are indicated. UNS, unstructured sequence. (Right) BSR cells were transfected with each of the indicated mRNAs, along with the RLUC transcript as an internal control. FLUC and RLUC activities were determined in cell lysates 8 h later, as indicated in Materials and Methods. Mean normalized FLUC values (FLUC/RLUC; means ± SD) from three independent experiments (each performed in triplicate) are shown as a percentage of data corresponding to 5′wt/3′wt_1 mRNA, taken as 100% (*, P < 0.05; **, P < 0.005).