Mc. Cassetti et al., DNA PACKAGING MUTANT - REPRESSION OF THE VACCINIA VIRUS A32 GENE RESULTS IN NONINFECTIOUS, DNA-DEFICIENT, SPHERICAL, ENVELOPED PARTICLES, Journal of virology, 72(7), 1998, pp. 5769-5780
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.