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FIG. 4.

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Genetic organization of the dicistronic replicon RNA, RibPVE2A(MluI), transcribed from the T7 RNA polymerase-based transcription construct pT7RibPVE2A(MluI). pT7RibPVE2A(MluI) encodes an RNA that contains the entire 5′ NCR and P1 coding region of PV separated from the P2/P3 coding regions, the 3′ NCR, and a poly(A) (>30 nt in length) by the EMCV IRES element. A cis-acting hammerhead ribozyme enzymatically cleaves itself (and the two guanosine residues of the T7 promoter) from the RNA, leaving the transcript with an authentic 5′ end. The 3′ terminus of the transcript contains four nonviral nucleotides imparted by the MluI restriction endonuclease site utilized for template linearization. The inserted fragment (see inset) encodes a UGA stop codon (Stop) followed by a 7-nt spacer, the EMCV IRES element (EMCV nt 260 to 834) followed by the first 15 nt of the EMCV open reading frame, and an additional tyrosine codon (Y) to provide a cis cleavage site for the generation of an authentic P2 N terminus following cleavage by the adjacent 2A proteinase (2Apro) activity. The initiation codon of the second cistron is indicated (M).

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