Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH 2 terminus of a gene encoding an Mr 62,000 polypeptide

Abstract

Marker transfer procedures were used to locate the site of mutation in the genome of a previously characterized ( B. Moss, E. N. Rosenblum, and P. Grimley, 1971), Virology 45, 135–148) rifampicin-resistant (Rif R) vaccinia virus isolate. Starting with a cosmid library prepared from the mutant genome, recombination with successively smaller DNA fragments was shown to transfer drug resistance to wild-type vaccinia virus. In this manner, the mutation was mapped within a 485-bp DNA segment in the central region of the genome at the extreme right end of the HindIII D fragment. Nucleotide sequencing indicated that this DNA segment differed from the homologous region of wild-type DNA by a single C/G → A/T substitution. Sequencing of the flanking 2195 by revealed two tandem nonoverlapping open reading frames (ORFs) encoding putative polypeptides of M r 16,908 and 61,840. The Rif R mutation resulted in a predicted glutamine → lysine change only 27 amino acids from the NH2 terminus of the longer ORF. A predicted asparagine to aspartic acid substitution, found in another Rif vaccinia virus mutant by 1. Tartaglia and E. Paoletti ( Virology 147, 394–404, 1985 ), mapped near the carboxyl terminus of the same ORF. These data suggest a model in which head-to-tail interaction between M r 61,840 polypeptides occurs and in which rifampicin blocks virus assembly by preventing this association.