Engineering recombinant poxviruses using a compact GFP–blasticidin resistance fusion gene for selection

Wong, Yik Chun; Lin, Chung-Cherng; Melo‐Silva, Carolina R.; Smith, Stewart A.; Tscharke, David C.

doi:10.1016/j.jviromet.2010.11.003

Cited by 29 publications

(30 citation statements)

References 20 publications

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“…1). WRD Left had 42 ORFs between VACWR001 (C23L) and VACWR043 (F4L) deleted and replaced with EGFP/bsd (blasticidin) (Wong et al, 2011) and VACWR021 (C7L); WRD Right had 25 ORFs between VACWR195 (B13R) and VACWR218 (B29R) replaced with mCherry. We maintained 3701 bp of non-coding DNA at the termini, including the essential concatemer resolution sequence, terminal hairpin and four blocks containing direct repeat elements (Merchlinsky & Moss, 1989;Merchlinsky, 1990;Garcia & Moss, 2001).…”

Section: Deletion Of 55 Orfs From the Genomic Termini Of Vacv Copenhamentioning

confidence: 99%

Redundancy complicates the definition of essential genes for vaccinia virus

Dobson

Tscharke

2015

Journal of General Virology

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Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants.

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Section: Deletion Of 55 Orfs From the Genomic Termini Of Vacv Copenhamentioning

confidence: 99%

Redundancy complicates the definition of essential genes for vaccinia virus

Dobson

Tscharke

2015

Journal of General Virology

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“…MVA-SIIN was made to match WR-SIIN and expresses the MSIINFEKL minigene under the control of p7.5 from TK of MVA. This virus was made for this study using a method based on transient dominant selection driven by a green fluorescent protein (GFP)-blasticidin selection marker (21). The TK targeting vector (pSC11GB) used had the GFP-blasticidin cassette from pSSGB (21) inserted into the HindIII site of the pSC11-SBAKN plasmid (22).…”

Section: Cells and Virusesmentioning

confidence: 99%

Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8⁺T Cells

Wong

Smith

Tscharke

2013

J Virol

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CD8؉ T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen. In this study, using two vaccinia virus (VACV) strains, namely, WR and MVA, we found that the CpG and cytc methods gave conflicting data. Moreover, we show for both strains of VACV that treatment of mice with CpG and cytc inhibited CD8 ؉ T cell responses to antigens designed to prime exclusively by direct presentation. Further investigation of the CpG method found that the extent to which priming is inhibited depends on the antigen examined, immunization route, replication ability of the virus, and, crucially, immunization dose. We suggest that greater caution is required when interpreting data using these methods and that priming pathways for antiviral CD8 ؉ T cells are not simply separated according to DC subsets or their maturation state.

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“…These intermediates can be resolved, following the removal of selection, to a virus with a tagged gene or back to the parental type. A similar method involving the use of both fluorescent and metabolic selection has been described previously 27 although the method used in this study utilizes shorter, synthesized regions of homology, allowing the identification of the desired recombinant virus by imaging the fluorescence of the tagged gene of interest. This secondary selection is only applicable for tagging highly expressed viral genes that produce sufficient fluorescence in a plaque assay.…”

Section: Discussionmentioning

confidence: 99%

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Marzook

Procter

Lynn

et al. 2014

JoVE

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Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

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Engineering recombinant poxviruses using a compact GFP–blasticidin resistance fusion gene for selection

Cited by 29 publications

References 20 publications

Redundancy complicates the definition of essential genes for vaccinia virus

Redundancy complicates the definition of essential genes for vaccinia virus

Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8⁺T Cells

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

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Engineering recombinant poxviruses using a compact GFP–blasticidin resistance fusion gene for selection

Cited by 29 publications

References 20 publications

Redundancy complicates the definition of essential genes for vaccinia virus

Redundancy complicates the definition of essential genes for vaccinia virus

Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8+T Cells

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

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Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8⁺T Cells