Vaccinia virus-encoded ribonucleotide reductase: sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants

The vaccinia virus gene that encodes the small subunit of ribonucleotide reductase was localized to the HindIII F fragment by using degenerate oligonucleotide probes. DNA sequencing revealed a leftward-reading open reading frame that predicted a protein of 37 kilodaltons whose amino acid sequence was much more homologous to the mouse and clam M2 sequences (approximately 80%) than to the corresponding herpesvirus (approximately 27%) or procaryotic (approximately 19%) gene products. Vaccinia virus mutants selected for the ability to grow in high concentrations of a specific inhibitor of ribonucleotide reductase, hydroxyurea, amplified the M2 gene and harbored tandem arrays (2 to 15 copies) of the gene within the HindIII F region. RNA isolated at early times after infection with wild-type virus and probed with an internal fragment of the M2 gene indicated one major (1.2 kilobases) and two minor (4.0 and 2.1 kilobases) transcripts. S1 nuclease analysis and primer extension experiments identified an RNA start site 12 nucleotides upstream of the putative initiation ATG codon.