Nucleotide sequence and transcriptional studies of the vaccinia virus KpnI I DNA fragment

The nucleotide sequence of the vaccinia virus (VV) KpnI I DNA fragment has been determined. This central, highly conserved portion of the VV genome corresponds to the right portion of the HindIII E, all of the HindIII O and P, and the left portion of the HindIII I DNA fragments. Computer-assisted analysis of this data indicated the presence of five tandemly oriented, leftward-reading open reading frames (ORFs) I-4, I-3, I-2, I-1, and O-1, with the I-4 ORF being an immediate early gene encoding the large M1 subunit of VV ribonucleotide reductase. Transcriptional analyses suggested that the I-3 and O-1 genes were constitutive genes, being expressed both before and after viral DNA synthesis. The I-1 and I-2 genes were late genes, expressed only after the initiation of viral DNA synthesis. Cell-free translation was used to confirm that the I-3, I-1, and O-1 ORFs were bonafide messages encoding proteins with molecular weights of 30, 35, and 71 kD, respectively. When the predicted amino acid sequences of the proteins encoded by the I-3, I-2, I-1, and O-1 genes were compared to the Genbank data base, no significant alignments were detected. Therefore, the biological functions of these proteins in the VV life cycle remain to be established.