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J Virol, July 1998, p. 5769-5780, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

Maria Cristina Cassetti,1,dagger Michael Merchlinsky,2 Elizabeth J. Wolffe,1 Andrea S. Weisberg,1 and Bernard Moss1,*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,1 and Laboratory of DNA Viruses, Center for Biologics Evaluation and Research, Food and Drug Administration,2 Bethesda, Maryland 20892

Received 30 January 1998/Accepted 2 April 1998

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-beta -D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.

* Corresponding author. Mailing address: Laboratory of Viral Diseases, NIAID, NIH, 4 Center Dr. MSC 0445, Bethesda, MD 20892-0445. Phone: (301) 496-9869. Fax: (301) 480-1147. E-mail: bmoss{at}nih.gov.

dagger Present address: Department of Molecular Biology, Rutgers University, Piscataway, NJ 08855-1179.

J Virol, July 1998, p. 5769-5780, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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