Genetic and molecular biological characterization of a vaccinia virus temperature-sensitive complementation group...

The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized. Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein. The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined. Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters. Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection. Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region. The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence. The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis. Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.