Assembly of virus particles during mixed infection with wild-type vaccinia and a rifampicin-resistant mutant

Abstract

A rifampicin-resistant vaccinia virus mutant was isolated from wild-type virus stocks. The mutant was purified by sucrose density gradient centrifugation and had an infectivity, electron microscopic appearance, and polypeptide composition that was similar to the wild-type virus. Growth of the mutant was inhibited only slightly at concentrations of rifampicin which completely prevented the growth of drug-sensitive virus, and at no concentration tested was there a detectable effect on viral envelope formation. The mutant virus was capable of rescuing wild-type during mixed infection in the presence of rifampicin; after simultaneous infection the ratio of genomes in the progeny reflected that of the original inoculum. Superinfection experiments demonstrated that beat-inactivated mutant was capable of efficiently rescuing wild-type virus and that extensive replication of the mutant genome was not required for this. Characteristic rifampicin blocked viral forms, normal maturing forms, and intermediate structures were seen in the same ultrathin cell sections after simultaneous infection with low multiplicities of mutant and wild-type virus. In cells simultaneously infected with high multiplicities of mutant and wild-type virus, rifampicin had no detectable effect on viral envelope formation. No difference was found in pulse-labeled polypeptides synthesized in rifampicin-treated cells infected with either mutant virus, wild-type virus, or both when analyzed by SDS-polyacrylamide gel electrophoresis. After a 4-hr chase, cleavage of the high molecular weight precursors of structural polypeptides was inhibited in cells infected with wild-type virus but occurred to similar extents in cells infected with mutant alone and cells simultaneously infected with mutant and wild-type virus.