DNA PACKAGING MUTANT - REPRESSION OF THE VACCINIA VIRUS A32 GENE RESULTS IN NONINFECTIOUS, DNA-DEFICIENT, SPHERICAL, ENVELOPED PARTICLES

The vaccinia virus A32 open reading frame was predicted to encode a pr otein with a nucleoside triphosphate-binding motif and a mass of 34 kD a, To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The re combinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-beta-D-thiogalactopyranosi de (IPTG), Under nonpermissive conditions, the mutant synthesized earl y-and late-stage viral proteins, as well as viral DNA that was process ed into unit-length genomes, Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature v irus particles but very few with nucleoids, Instead of brick-shaped ma ture particles with defined core structures, there were numerous elect ron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extr acellular enveloped virions. Mutant viral particles, purified by sucro se density gradient centrifugation, had low infectivity and transcript ional activity, and the majority were spherical and lacked DNA, Nevert heless, the particle preparation contained representative membrane pro teins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that in corporation of these components is not strictly coupled to DNA packagi ng.