Vaccinia virus rifampicin-resistance locus specifies a late 63,000 Da gene product

The genetic locus specifying rifampicin-resistance (RifR) in a vaccinia virus mutant has been localized by marker rescue analysis (J. Tartaglia and E. Paoletti (1985) Virology 147, 394-404). The mutation was defined by DNA sequence analysis as an AT to GC transition occurring 56 bp to the left of the unique XhoI site within HindIII D. The point mutation resulted in an asparagine to aspartic acid substitution 60 amino acids from the predicted C-terminus. Specific DNA probes were used to characterize the RifR designated gene at the transcriptional and translational levels. This region is transcriptionally active only after vaccinia virus DNA synthesis, but not in the presence of cytosine arabinoside suggesting that the RifR function is a late gene product. Translation of hybrid selected RNA to DNA surrounding the mutant marker directed the synthesis of a polypeptide with an apparent mol wt of 63 kDa. Transcriptional and translational mapping studies showed that the mRNA encoding this 63-kDa polypeptide was initiated approximately 460 bp to the right of the HindIII D-A junction and was transcribed in a leftward direction into the HindIII D region.