FIG 1.

Schematic representation of the wild-type MVEV genome and two genetically engineered, bicistronic MVEV-based constructs. (A) MVEV.wt has a single, long open reading frame encoding all structural (C, prM, and E) and nonstructural (NS1 to NS5) proteins and flanked by untranslated regions (5′ and 3′ UTR). (B) For construction of MVEV.C-IRES, an opal stop codon (filled triangle) was introduced at the C terminus of C protein, followed by an EMCV IRES, which drives translation of all other viral proteins, starting at the signal peptide of prM (black box). (C) Construct MVEV.IFN-β has the mouse IFN-β gene (blue box) inserted downstream of the prM signal peptide, which directs the nascent IFN protein into the lumen of the ER; the IFN-β gene is flanked at the 3′ end by an opal stop codon (filled triangle), followed by EMCV IRES sequence (yellow arrow). The amino acid sequence (single-letter code) and corresponding nucleotide sequence of the prM signal peptide are shown at the bottom (60). Note that synonymous nucleotide changes were introduced into the prM signal peptide upstream of the IRES in construct MVEV.IFN-β to prevent homologous recombination and deletion of the foreign sequences from the viral genome (see sequence alignment; the top and bottom nucleotide sequences encode the wild-type and the altered prM signal peptide, respectively; the resulting amino acid sequence is in uppercase letters, and dots represent identical residues).