Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus

Abstract

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40؇C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [ 3 H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.

Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly

“…5, right). Such abnormal half-moon forms were described on April 30, 2020 by guest http://jvi.asm.org/ previously for cells infected at the restrictive temperature with a temperature-sensitive vaccinia virus bearing a mutation in the I7 protein (25), the cysteine protease responsible for processing the major virion structural protein precursors (1,6,7,18,33).…”

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

“…Important and still unresolved issues about VV assembly are how the full complement of proteins become wrapped by the viral membrane to generate individual spherical IVs and how the genomic DNA is incorporated into these particles. Several studies suggested that the viral DNA packed into a nucleoprotein complex is taken up before the IVs are completely sealed (13,18,28,31).…”

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

“…Previous studies have suggested that during infection with wild-type virus, the nucleoids enter IV just before the membrane is sealed (13,19,31,34). Evidence that such nucleoids contain DNA has been obtained by using [ 3 H]thymidine labeling (19) and immunoelectron microscopy (13). In the absence of IPTG, the low number of IV with nucleoids suggested that the A32 protein is needed for DNA packaging.…”

“…The I7 ORF encodes a structural protein with some sequence similarity to the type II topoisomerase of Saccharomyces cerevisiae (22). At nonpermissive temperatures, morphogenesis of an I7 ts mutant was interrupted at a stage between IV and IMV, with the accumulation of dense, spherical particles (9,13,22). The I7 mutant particles, however, contain nucleoids and DNA, indicating that morphogenesis was arrested at a slightly later stage than that which occurred with vA32i.…”