Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

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High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells

Journal of Virology ◽

10.1128/jvi.77.13.7281-7290.2003 ◽

2003 ◽

Vol 77 (13) ◽

pp. 7281-7290 ◽

Cited By ~ 26

Author(s):

Xiao-Dan Yao ◽

David H. Evans

Keyword(s):

Vaccinia Virus ◽

Genetic Recombination ◽

Virus Genome ◽

Dna Fragments ◽

Gene Encoding ◽

Plaque Purification ◽

Infected Cells ◽

Clone And Express ◽

Recombination Reactions ◽

Multiple Pcr

ABSTRACT Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 106 PFU/μg of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.

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A Temperature-Sensitive Mutation of the Vaccinia Virus E11 Gene Encoding a 15-kDa Virion Component

Virology ◽

10.1006/viro.1996.0057 ◽

1996 ◽

Vol 216 (1) ◽

pp. 252-257 ◽

Cited By ~ 9

Author(s):

SHUANG PING WANG ◽

STEWART SHUMAN

Keyword(s):

Vaccinia Virus ◽

Temperature Sensitive ◽

Gene Encoding ◽

Temperature Sensitive Mutation

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Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity

Journal of General Virology ◽

10.1099/vir.0.055921-0 ◽

2013 ◽

Vol 94 (11) ◽

pp. 2367-2392 ◽

Cited By ~ 185

Author(s):

Geoffrey L. Smith ◽

Camilla T. O. Benfield ◽

Carlos Maluquer de Motes ◽

Michela Mazzon ◽

Stuart W. J. Ember ◽

...

Keyword(s):

Immune Response ◽

Vaccinia Virus ◽

Innate Immune Response ◽

Immune Evasion ◽

Mammalian Cells ◽

Innate Immune ◽

Gene Encoding ◽

Cytokines And Chemokines ◽

Host Innate Immune Response ◽

New Hosts

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

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