Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription

Fonseca, Francine Souza Alves da; Moss, Bernard

doi:10.1073/pnas.1534874100

Cited by 44 publications

(37 citation statements)

References 36 publications

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“…We considered that if the putative D10 deletion mutant was replication-impaired, then the toxic effect of Geniticin may have prevented the formation of visible plaques in previous attempts. A similar problem in isolating a VACV topoisomerase mutant was overcome by avoiding drug selec- tion and using EGFP as an insertion marker (12). When we tried this alternative strategy, we were able to find small green fluorescent plaques formed by the D10R deletion mutant.…”

Section: Discussionmentioning

confidence: 99%

“…D10R is one of only a few genes conserved in all sequenced poxviruses that have been shown to be nonessential for replication in tissue culture cells. Another nonessential conserved gene is the type 1 topoisomerase (12). Although present in all chordopoxviruses, D9R is absent from entomopoxviruses.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, D10R is one of the few genes conserved in all sequenced poxviruses that have been shown to be nonessential for replication in tissue culture cells. Another example is the type 1 topoisomerase (12). Nevertheless, replication of the D10R deletion mutant virus was impaired, as it made small plaques on cell monolayers and the specific infectivity of virus particles was reduced by 6 to 10 fold.…”

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confidence: 99%

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Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

Parrish

Moss

2006

J Virol

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103

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The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (v⌬D10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (v⌬D9). Purified v⌬D10 virions appeared normal by microscopic examination and biochemical analysis but produced 6-to 10-fold-fewer plaques at the same concentration as wild-type or v⌬D9 virions. When 4 PFU per cell of wild-type or v⌬D9 virions or equal numbers of v⌬D10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in v⌬D10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in v⌬D10-infected cells than in cells infected with the wild type or v⌬D9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in v⌬D10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.Vaccinia virus (VACV), the most extensively studied member of the Poxviridae family, contains a large double-stranded DNA genome encoding approximately 200 proteins. Replication occurs in the cytoplasm and is orchestrated in a programmed manner, with sequential transcription of early-, intermediate-, and late-stage genes (25). Numerous positive regulatory factors have been identified, notably the proteins required for stage-specific transcription in conjunction with the viral DNA-dependent RNA polymerase (for a review, see reference 8). The changes in total viral early, intermediate, and late RNA levels, however, suggest the deployment of negative, as well as positive, regulators (3). Indeed, the speed at which RNA levels change can only be explained if viral RNAs have very short half-lives, as was proposed nearly 40 years ago (26, 31). The accelerated turnover of cellular mRNAs during VACV infection (9,19,20,29) might occur by a related or identical degradation mechanism.A putative negative regulator of gene expression was identified in a transfection-based DNA library screen that was successfully used to find activators of VACV late transcription (21, 32). The inhibitory activity was mapped primarily to the D10R gene of VACV, with the D9R gene contributing more modestly to the effect. (Note: VACV genes are designated with a capital letter that describes the HindIII fragment containing the open reading frame, followed by a number that corresp...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

Parrish

Moss

2006

J Virol

Self Cite

103

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“…Plasmid orientation of GAD under the control of vaccinia P7.5 promoter was confirmed by DNA sequencing. Recombinant virus was generated as described elsewhere (26). Briefly, VV (Western Reserve)-infected TK Ϫ 143B cells were transfected with plasmid pSC11GAD containing the mutant GAD flanked by the VV TK sequence to provide sites for homologous recombination.…”

Section: Generation Of Rvv-expressing Gad Proteinmentioning

confidence: 99%

Disruption of MHC Class II-Restricted Antigen Presentation by Vaccinia Virus

Wang

Zhou

et al. 2005

The Journal of Immunology

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Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.

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“…Vaccinia viruses lacking the gene encoding this enzyme are still viable but exhibit early-gene-transcription defects (7). This suggests that poxvirus topoisomerases serve a biological function resembling that of cellular Topo I.…”

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confidence: 99%

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Lin¹,

Li²,

Irwin³

et al. 2008

J Virol

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Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drugresistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase II␤. One can prevent the interaction by introducing a C 11 -to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase II␣/␤ in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase II␣ and II␤ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases II␣/␤ in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.Topoisomerases play a critical role in modulating DNA superhelical density and in unknotting and decatenating the structures formed during replication, recombination, and repair (reviewed in references 2 and 5). Mammalian cells carry several different kinds of topoisomerases, classified as being either type I or type II enzymes. Both classes of enzyme catalyze DNA strand breakage and rejoining, but type I enzymes catalyze reactions involving single-strand breaks, while the type II topoisomerases catalyze double-strand cleavage reactions. The mammalian type IB enzyme (Topo I) is well adapted for removing the superhelical stress created by the replication and transcription machinery. The two closely related type IIA enzymes (Topo II␣ and Topo II␤) may also catalyze these same reactions but probably play a more critical role in processes like chromatin reorganization and chromosome segregation. A third class of type IA enzymes (Topo III␣ and Topo III␤)

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Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription

Cited by 44 publications

References 36 publications

Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

Disruption of MHC Class II-Restricted Antigen Presentation by Vaccinia Virus

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

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