Cloning the vaccinia virus genome as a bacterial artificial chromosome in
            <i>Escherichia coli</i>
            and recovery of infectious virus in mammalian cells

Domi, Arban; Moss, Bernard

doi:10.1073/pnas.192420599

Cited by 96 publications

(83 citation statements)

References 35 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Only recently has the cloning of large-DNA viruses as infectious clones in bacteria been successful (1,12,22). Here, we present a comprehensive mutagenesis procedure that is generally applicable to any gene of a large cloned viral genome.…”

Section: Vol 78 2004mentioning

confidence: 99%

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication

Bubeck

Wagner

Ruzsics

et al. 2004

J Virol

124

View full text Add to dashboard Cite

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.Cytomegaloviruses define the beta subgroup of the Herpesviridae, which are important animal and human pathogens. Human cytomegalovirus (HCMV) is detected with a high prevalence in the human population, and it causes severe and fatal disease in immunocompromised individuals. Murine cytomegalovirus (MCMV) infection serves as an animal model system. CMVs harbor large double-stranded DNA genomes of about 230 kbp (7, 32). The recent cloning of the HCMV and MCMV genomes as infectious bacterial artificial chromosomes (BACs) (4,15,24,27,45,50) rendered these genomes accessible to advanced genetic manipulation (for a review, see reference 46).Infectious herpesvirus particles are formed during lytic virus replication as a result of a complex maturation and egress process (for a review, see reference 28). This multistep process starts in the nucleus with the packaging of viral genomes into capsids. The final envelopment takes place in the trans-Golgi network. Each maturation step is controlled by particular multiprotein assemblies, which are formed by complex interactions between viral and cellular proteins. Efforts to reconstitute the first morphogenesis stage, the capsid assembly, have been successful for alphaherpesviruses (43). In the mature virion, some higher-order protein complexes were studied by crystallography and electron microscopy (for a review, see reference 9). Yet, the logistics of the dynamic transitory stages (e.g., capsid egress from the nucleus) are not accessible for high-resolution analysis using static physical methods. Artificial reconstitution of these intermediate stages would require regulated coexpression of a number of viral proteins in the context of an appropriate host cell environment. Thus, detailed structure-function analyses of protein machines, which are associated with nuclear egress of virus capsids, need the context of virus replication. Gene products involved in herpesvirus morphogenesis are often essential for viral growth. The genetic analysis of these genes is restricted to either the isolated expression of the protein in cell ...

show abstract

Section: Vol 78 2004mentioning

confidence: 99%

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication

Bubeck

Wagner

Ruzsics

et al. 2004

J Virol

124

View full text Add to dashboard Cite

show abstract

“…transfected into CV-1 cells that had been infected with 0.1 PFU per cell of helper fowlpox virus as described previously (9). After 7 days, cells were harvested, and the lysate was applied to BS-C-1 cells and covered with a solid agar overlay.…”

mentioning

confidence: 99%

Vaccinia Virus E2L Null Mutants Exhibit a Major Reduction in Extracellular Virion Formation and Virus Spread

Domi

Weisberg

Moss

2008

J Virol

Self Cite

View full text Add to dashboard Cite

The vaccinia virus E2L (VACWR058) gene is conserved in all sequenced chordopoxviruses and is predicted to encode an 86-kDa protein with no recognizable functional motifs or nonpoxvirus homologs. Although the region immediately upstream of the open reading frame lacked optimal consensus promoter motifs, expression of the E2 protein occurred after viral DNA replication. Transfection studies, however, indicated that the promoter was weak compared to well-characterized intermediate and late promoters. The E2 protein was present in mature virions purified from infected cells but was more abundant in extracellular enveloped forms. Despite the conservation of the E2L gene in chordopoxviruses, deletion mutants could be isolated from both the WR and IHD-J strains of vaccinia virus. These null mutants produced very small plaques in all cell lines tested, reduced amounts of mature infectious virions, and very low numbers of extracellular virions. Nevertheless, viral protein synthesis appeared qualitatively and quantitatively normal. The defect in extracellular virus formation was corroborated by electron microscopy, which also showed some aberration in the wrapping of virions by cisternal membranes. Extracellular virions that did form, however, were able to induce actin tail formation.Poxviruses are large, enveloped, double-stranded DNA viruses that replicate entirely within the cytoplasm of vertebrate or invertebrate cells. The most intensively studied member of the family, vaccinia virus (VACV), contains approximately 200 genes, of which nearly half are conserved in all chordopoxviruses (37). The highly conserved genes encode proteins with roles in viral transcription, genome replication, and the formation of progeny virus particles (22). The viral proteins required for mRNA synthesis and modification include a multisubunit DNA-dependent RNA polymerase; early, intermediate, and late stage-specific transcription factors; elongation factors; capping and methylating enzymes; and a poly(A) polymerase. At least six viral proteins are needed for replication and processing of the viral genome (23), and many more are involved in assembly of the infectious virus particle (6). The latter include membrane proteins, components of the core, a redox system, and proteinases. Many of the less highly conserved proteins are nonessential for replication in tissue culture and have host response functions, including immune evasion (24).Our present ignorance of the roles of many conserved genes hampers research into the reproductive cycle of poxviruses. Fortunately, experimental methods which can improve this situation are available. The purpose of the present study was to carry out the initial characterization of the product of the VACWR058 (E2L according to the Copenhagen strain nomenclature) open reading frame (ORF), which is conserved in all sequenced chordopoxviruses. We found that the encoded protein (referred to as E2) was expressed postreplicatively and was detected in purified extracellular enveloped virions (EVs) and to a lesser ext...

show abstract

“…The 139-kb maize chloroplast genome has been cloned in yeast (5), and the 135-kb rice chloroplast genome has been cloned in B. subtilis (1). Viral genomes have been cloned in E. coli, including those from a number of herpesviruses (6) as well as baculoviruses (7)(8)(9), coronaviruses (10) and a poxvirus (11). A 36-kb human adenovirus type 2 genome (12) and a 178-kb fragment of the human cytomegalovirus genome (13) have been cloned in yeast.…”

Section: Introductionmentioning

confidence: 99%

Cloning Whole Bacterial Genomes in Yeast

Benders

2012

Methods in Molecular Biology

View full text Add to dashboard Cite

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

show abstract

Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells

Cited by 96 publications

References 35 publications

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication

Vaccinia Virus E2L Null Mutants Exhibit a Major Reduction in Extracellular Virion Formation and Virus Spread

Cloning Whole Bacterial Genomes in Yeast

Contact Info

Product

Resources

About