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DAXX modulates human papillomavirus early gene expression and genome replication in U2OS cells.

Kivipõld P, Võsa L, Ustav M, Kurg R - Virol. J. (2015)

Bottom Line: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells.In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technology, University of Tartu, Nooruse 1, 50411, Tartu, Estonia. piia.kivipold@gmail.com.

ABSTRACT

Background: The human papillomavirus (HPV) genomes can replicate, and are maintained as autonomously replicating extrachromosomal plasmids in human U2OS cells. Previous studies have shown that HPV genomes are transcriptionally active in U2OS cells and can express the viral early proteins required for initiation and establishment of HPV replication. In the present work, we have examined the involvement of cellular DAXX protein in HPV replication in U2OS cells.

Methods: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells. In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.

Results: We show that a portion of HPV replication foci are partially co-localized with components of ND10, cellular DAXX and PML proteins. In addition, we demonstrate that the knock-down of the cellular DAXX protein modulates the HPV genome replication and transcription in U2OS cells--papillomavirus replication is reduced in the absence of this component of ND10.

Conclusions: The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

No MeSH data available.


Related in: MedlinePlus

Down-regulation of DAXX represses HPV genome transient replication. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 (a) or wt HPV11 (b) genome. Cells were harvested 72 h after transfection, episomal DNA was isolated, digested with DpnI and linearizing enzyme, EcoRI or BamHI, and analyzed by Southern blotting using a radiolabeled probe specific for the viral genome. The linearizing enzyme (M1) and DpnI (M2) digested pBRHPV18 or pUCHPV11 plasmids as markers are shown on the right. c qPCR analysis of the episomal DNA isolated in replication assays. Episomal DNA was digested with DpnI and analysed by qPCR using HPV18 or HPV11 genome, and mitochondrial DNA specific primers. The data represent the average of three independent experiments and are presented graphically relative to the basal replication level of the HPV18 or HPV11 genome. d Western blot analysis of the DAXX protein levels in the cells used in replication assays. Cells (105) were lysed, separated electrophoretically, and immunoblotted with anti-DAXX and anti-tubulin antibodies
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Fig4: Down-regulation of DAXX represses HPV genome transient replication. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 (a) or wt HPV11 (b) genome. Cells were harvested 72 h after transfection, episomal DNA was isolated, digested with DpnI and linearizing enzyme, EcoRI or BamHI, and analyzed by Southern blotting using a radiolabeled probe specific for the viral genome. The linearizing enzyme (M1) and DpnI (M2) digested pBRHPV18 or pUCHPV11 plasmids as markers are shown on the right. c qPCR analysis of the episomal DNA isolated in replication assays. Episomal DNA was digested with DpnI and analysed by qPCR using HPV18 or HPV11 genome, and mitochondrial DNA specific primers. The data represent the average of three independent experiments and are presented graphically relative to the basal replication level of the HPV18 or HPV11 genome. d Western blot analysis of the DAXX protein levels in the cells used in replication assays. Cells (105) were lysed, separated electrophoretically, and immunoblotted with anti-DAXX and anti-tubulin antibodies

Mentions: In order to analyze the involvement of the DAXX protein in HPV genome replication in U2OS cells, we knocked down the expression of DAXX with siRNA and transfected the cells with wt HPV11 and wt HPV18 genomic DNA. Episomal DNA was isolated 72 h after transfection, and the newly replicated HPV11 and HPV18 viral DNA was analyzed by Southern blot (Fig 4a and b). Down-regulation of DAXX repressed the initial replication of both HPV11 and HPV18 genomes. We also analyzed the HPV DNA levels by quantitative real-time PCR, using mitochondrial DNA as an internal control. Consistent with Southern blot analysis, the quantitation of replication products by real-time PCR indicated that down-regulation of the DAXX protein reduced HPV DNA replication by 2-3-fold (Fig. 4c). The level of DAXX protein in U2OS cells treated with siDaxx or siCtr is shown in Fig. 4d.Fig. 4


DAXX modulates human papillomavirus early gene expression and genome replication in U2OS cells.

Kivipõld P, Võsa L, Ustav M, Kurg R - Virol. J. (2015)

Down-regulation of DAXX represses HPV genome transient replication. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 (a) or wt HPV11 (b) genome. Cells were harvested 72 h after transfection, episomal DNA was isolated, digested with DpnI and linearizing enzyme, EcoRI or BamHI, and analyzed by Southern blotting using a radiolabeled probe specific for the viral genome. The linearizing enzyme (M1) and DpnI (M2) digested pBRHPV18 or pUCHPV11 plasmids as markers are shown on the right. c qPCR analysis of the episomal DNA isolated in replication assays. Episomal DNA was digested with DpnI and analysed by qPCR using HPV18 or HPV11 genome, and mitochondrial DNA specific primers. The data represent the average of three independent experiments and are presented graphically relative to the basal replication level of the HPV18 or HPV11 genome. d Western blot analysis of the DAXX protein levels in the cells used in replication assays. Cells (105) were lysed, separated electrophoretically, and immunoblotted with anti-DAXX and anti-tubulin antibodies
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492069&req=5

Fig4: Down-regulation of DAXX represses HPV genome transient replication. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 (a) or wt HPV11 (b) genome. Cells were harvested 72 h after transfection, episomal DNA was isolated, digested with DpnI and linearizing enzyme, EcoRI or BamHI, and analyzed by Southern blotting using a radiolabeled probe specific for the viral genome. The linearizing enzyme (M1) and DpnI (M2) digested pBRHPV18 or pUCHPV11 plasmids as markers are shown on the right. c qPCR analysis of the episomal DNA isolated in replication assays. Episomal DNA was digested with DpnI and analysed by qPCR using HPV18 or HPV11 genome, and mitochondrial DNA specific primers. The data represent the average of three independent experiments and are presented graphically relative to the basal replication level of the HPV18 or HPV11 genome. d Western blot analysis of the DAXX protein levels in the cells used in replication assays. Cells (105) were lysed, separated electrophoretically, and immunoblotted with anti-DAXX and anti-tubulin antibodies
Mentions: In order to analyze the involvement of the DAXX protein in HPV genome replication in U2OS cells, we knocked down the expression of DAXX with siRNA and transfected the cells with wt HPV11 and wt HPV18 genomic DNA. Episomal DNA was isolated 72 h after transfection, and the newly replicated HPV11 and HPV18 viral DNA was analyzed by Southern blot (Fig 4a and b). Down-regulation of DAXX repressed the initial replication of both HPV11 and HPV18 genomes. We also analyzed the HPV DNA levels by quantitative real-time PCR, using mitochondrial DNA as an internal control. Consistent with Southern blot analysis, the quantitation of replication products by real-time PCR indicated that down-regulation of the DAXX protein reduced HPV DNA replication by 2-3-fold (Fig. 4c). The level of DAXX protein in U2OS cells treated with siDaxx or siCtr is shown in Fig. 4d.Fig. 4

Bottom Line: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells.In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technology, University of Tartu, Nooruse 1, 50411, Tartu, Estonia. piia.kivipold@gmail.com.

ABSTRACT

Background: The human papillomavirus (HPV) genomes can replicate, and are maintained as autonomously replicating extrachromosomal plasmids in human U2OS cells. Previous studies have shown that HPV genomes are transcriptionally active in U2OS cells and can express the viral early proteins required for initiation and establishment of HPV replication. In the present work, we have examined the involvement of cellular DAXX protein in HPV replication in U2OS cells.

Methods: We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells. In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.

Results: We show that a portion of HPV replication foci are partially co-localized with components of ND10, cellular DAXX and PML proteins. In addition, we demonstrate that the knock-down of the cellular DAXX protein modulates the HPV genome replication and transcription in U2OS cells--papillomavirus replication is reduced in the absence of this component of ND10.

Conclusions: The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.

No MeSH data available.


Related in: MedlinePlus