Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes

Zhang, Xu; Zikos, Dimitrios; Osterrieder, Nikolaus; Tischer, B. Karsten

doi:10.1128/jvi.02385-13

Cited by 17 publications

(21 citation statements)

References 45 publications

(50 reference statements)

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“…A BAC clone of CPXV-BR containing monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), driven by early and late poxviral promoters, respectively, in the mini-F region, was generated previously and termed pBRFseR (10). Virus reconstituted from pBRFseR was used as the wildtype control in this study and termed vBRFseR.…”

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

“…Single-gene deletion mutants of CPXV-BR were reconstituted from mutant bacterial artificial chromosomes (BACs) previously (10). All CPXVs used in this study were propagated and titrated on Vero cells.…”

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

“…Virus reconstituted from pBRFseR was used as the wildtype control in this study and termed vBRFseR. Clones of a library of targeted knockout CPXV-BR BAC mutants for each single viral ORF (10) were maintained in Escherichia coli strain GS1783 (12). BAC clones were modified by two-step Red-mediated recombination (12, 13) using marker constructs containing kanamycin, tetracycline, or ampicillin resistance genes.…”

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

“…Glass beads with a diameter of 0.75 to 1 mm (Karl Roth, Karlsruhe, Germany) were added into the tube and vortexed for 90 s before the cell suspension was centrifuged for 15 min at 300 rpm. The supernatant was collected and stored at Ϫ70°C, and titers were determined by plaque assay of 10-fold serial dilutions on Vero cells (10).…”

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

“…Recently, we generated a library of targeted knockout CPXV strain Brighton (CPXV-BR) mutants for each single viral open reading frame (ORF) (10) as well as a construct with a deletion of all six genes encoding kelch-like proteins. Reconstitution of infectious virus was successful for 109 of the 183 singlegene deletion mutants as well as for the mutant devoid of kelchlike genes.…”

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confidence: 99%

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Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

Zhang

Zikos

Tamošiūnaitė³

et al. 2014

J Virol

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Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown. IMPORTANCE It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virusCrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.

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Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

Section: Cell Lines African Green Monkey Cells Vero 76 (Collection Omentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

Zhang

Zikos

Tamošiūnaitė³

et al. 2014

J Virol

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A Review on Graph Analytics-Based Approaches in Protein-Protein Interaction Network

Narmadha

Pravin

Sundar

et al. 2020

Proceedings of International Conference on Artificial Intelligence, Smart Grid and Smart City Applications

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Polyglutamine Repeats in Viruses

Schein

2018

Mol Neurobiol

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This review explores the presence and functions of polyglutamine (polyQ) in viral proteins. In mammals, mutations in polyQ segments (and CAG repeats at the nucleotide level) have been linked to neural disorders and ataxias. PolyQ regions in normal human proteins have documented functional roles, in transcription factors and, more recently, in regulating autophagy. Despite the high frequency of polyQ repeats in eukaryotic genomes, little attention has been given to the presence or possible role of polyQ sequences in virus genomes. A survey described here revealed that polyQ repeats occur rarely in RNA viruses, suggesting that they have detrimental effects on virus replication at the nucleotide or protein level. However, there have been sporadic reports of polyQ segments in potyviruses and in reptilian nidoviruses (among the largest RNA viruses known). Conserved polyQ segments are found in the regulatory control proteins of many DNA viruses. Variable length polyQ tracts are found in proteins that contribute to transmissibility (cowpox A-type inclusion protein (ATI)) and control of latency (herpes viruses). New longer-read sequencing methods, using original biological samples, should reveal more details on the presence and functional role of polyQ in viruses, as well as the nucleotide regions that encode them. Given the known toxic effects of polyQ repeats, the role of these segments in neurovirulent and tumorigenic viruses should be further explored.

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Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes

Abstract: Cowpox virus (CPXV) belongs to the genus

Cited by 17 publications

References 45 publications

Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes

A Review on Graph Analytics-Based Approaches in Protein-Protein Interaction Network

Polyglutamine Repeats in Viruses

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