Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA.

Abstract

MATERIALS AND METHODSCells and Viruses. FEL cells (a subclone of line 745A) were grown in Dulbecco's modified Eagle's medium (DME medium) containing 10% fetal calf serum and antibiotics. These cells have been previously used to study antiviral and interferon-mediated enzyme activities during infection with vaccinia virus (3). The plaque-purified WR strain of vaccinia virus was grown in HeLa S3 spinner cells at a multiplicity of 0.1 plaque-forming unit (pfu) per cell and purified essentially according to the method of Joklik (4). The particle-to-pfu ratio was about 27 when titrated on monolayer cultures of monkey BSC-40 cells. FEL cells were infected with purified vaccinia virus at 1.0 pfu per cell. On the second and third day ofinfection >90% of the cells had died and remained attached to the dish. Surviving cells were removed by centrifugation and resuspended in fresh medium. After recovery (2-3 weeks) the cells were serially passaged every 4-5 days.Characterization of Intracellular DNA and of Virion DNA.Conditions for extraction of high molecular weight DNA from whole cell lysates, isolation of WR-DNA from purified virus, digestion of DNA with restriction endonucleases, gel electrophoresis, Southern blotting, and hybridization with specific nick-translated DNA probes have been previously described (5, 6). Virus from persistently infected cells was plaque purified by selection of well-isolated plaques on monolayers of BSC-40 cells. Independent virus isolates were propagated in BSC-40 cells and virus was purified as described (7). Where appropriate, these viruses were labeled in the DNA with [32P]orthophosphate or [3H]thymidine (7).

Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence

“…This will be the subject of further research. The presence of long stretches of non-essential DNA near the two ends of the genome is still an unexplained structural feature of the orthopoxviruses (Gangemi & Sharp, 1976;Panicali et al, 1981;Paez et al, 1985). It is assumed that these sequences encode a variety of proteins which interact with the host (Moyer et al, 1980;Moss et al, 1981;Pickup et al, 1986;Kotwal & Moss, 1988;Smith et al, 1989).…”

Evolution of and Evolutionary Relationships between Extant Vaccinia Virus Strains

“…Comprehensive analyses of these genomic changes that lead to gene truncation and fragmentation can sometimes detect remnants of the evolutionary process still present in orthopoxvirus genomes (14). Large deletions and other kinds of genome rearrangements are also commonly detected (15,16), especially around the terminal inverted repeats (TIRs) (17), and can significantly affect the lengths of VACV genomes. They can also alter the number of duplicated versus unique genes and can be associated with reduced infectivity and pathogenicity (16,17).…”

“…Large deletions and other kinds of genome rearrangements are also commonly detected (15,16), especially around the terminal inverted repeats (TIRs) (17), and can significantly affect the lengths of VACV genomes. They can also alter the number of duplicated versus unique genes and can be associated with reduced infectivity and pathogenicity (16,17). Because these larger rearrangements are less common and not readily obscured by sequence drift, they provide a more useful tool than SNPs for studying deeper evolutionary relationships between viruses.…”

Vaccinia virus glycoproteins and immune evasion: The Sixteenth Fleming Lecture

“…The vaccinia virus gene(s) encoding the protein(s) mediating resistance to IFN are localized near the left ITR since virus replication in the presence of IFN prevents the spontaneous deletion of DNA from this region (Paez & Esteban, 1985). A factor responsible for the rescue of VSV is a virus-specified early protein that inhibits the P1 protein kinase-mediated phosphorylation of the eIF2c~ (Whitaker-Dowling & Youngner, 1983Paez & Esteban, 1984b;.…”