Molecular construction of the DEN1 chimeric cDNA plasmids. A. The CME structural protein coding region of the DEN4 cDNA plasmid p4 was replaced with the corresponding region from DEN1 (Puerto Rico/94) CME sub-clone pUCBXR-init. Following introduction of a Pst I site near the C-M junction of pUCBXR-init, the ME region of p4 was replaced with the corresponding region from DEN1. Restriction sites used to facilitate the construction are indicated. The virus genomic regions for each of the resulting full-length chimeric cDNA plasmids are shown and the presence of the Δ30 mutation is indicated in the 3'-UTR. DEN1 sequence is shaded. The resulting cloning junctions are numbered 1 – 5 and the inserted linker sequence is indicated as a hatched box. The location of the linker is indicated. B. The nucleotide sequences surrounding the cloning junctions identified above are shown along with the predicted amino acid sequence corresponding to the virus polyprotein. Lower case letters indicate nucleotide substitutions that differ from wild type. For junction 3, the amino acid sequence is shown as predicted for the virus genome (with the linker sequence removed prior to transcription) and as predicted in E. coli where cryptic expression of the virus polyprotein with the linker sequence intact would result in translational termination. The termination codon in the polyprotein open reading frame that is located in the linker is indicated by * * *. Termination codons exist in the linker sequence for each of the additional forward and reverse open reading frames. For junction 4, the introduced Pst I site is indicated. C = capsid protein; M = membrane protein (with precursor region), E = envelope protein; NS = nonstructural.