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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

HPV replication foci in U2OS cells. Immunofluorescence analysis of U2OS cells transfected with HPV11 genome. U2OS cells were transfected with wt HPV11 minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV11 E2 (green) and BRD4 (red). Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. a and b represent cells with different number and size of HPV11 E2 foci. On the right panels, the intensity profiles are shown
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Fig1: HPV replication foci in U2OS cells. Immunofluorescence analysis of U2OS cells transfected with HPV11 genome. U2OS cells were transfected with wt HPV11 minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV11 E2 (green) and BRD4 (red). Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. a and b represent cells with different number and size of HPV11 E2 foci. On the right panels, the intensity profiles are shown

Mentions: First, we analyzed the involvement of cellular BRD4 protein in HPV replication in U2OS cells. BRD4 localizes to, and is essential for the formation of HPV replication foci in primary human foreskin keratinocytes. However, when E1 and E2 begin to amplify the viral DNA, BRD4 is displaced from the foci [21]. In order to test whether this is also the case in U2OS cells, we performed an immunofluorescence assay. Six days after transfection with 2 μg of wt HPV11 minicircle genomes, the cells were fixed and immunostained for HPV11 E2 and BRD4 proteins. As shown in Fig. 1, the individual U2OS cells contained different number of HPV11 E2 foci (Fig. 1 HPV11 E2 column). We detected up to ten HPV11 E2 foci, with an average value of four foci per cell nucleus. The HPV replication foci also had a different size. The small HPV11 E2 foci co-localized with the cellular BRD4 protein (Fig. 1a), and were reminiscent of the small early HPV replication foci that were described in human keratinocytes by Sakakibara and others [21]. Large HPV11 E2 foci were also visible in U2OS cells at the same time-point, and the cellular BRD4 protein was absent from these foci (Fig. 1b). Overall, this indicates the involvement of BRD4 in the early steps of replication foci formation in U2OS cells, similar to what has been observed in human keratinocytes [21].Fig. 1


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

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

HPV replication foci in U2OS cells. Immunofluorescence analysis of U2OS cells transfected with HPV11 genome. U2OS cells were transfected with wt HPV11 minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV11 E2 (green) and BRD4 (red). Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. a and b represent cells with different number and size of HPV11 E2 foci. On the right panels, the intensity profiles are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: HPV replication foci in U2OS cells. Immunofluorescence analysis of U2OS cells transfected with HPV11 genome. U2OS cells were transfected with wt HPV11 minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV11 E2 (green) and BRD4 (red). Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. a and b represent cells with different number and size of HPV11 E2 foci. On the right panels, the intensity profiles are shown
Mentions: First, we analyzed the involvement of cellular BRD4 protein in HPV replication in U2OS cells. BRD4 localizes to, and is essential for the formation of HPV replication foci in primary human foreskin keratinocytes. However, when E1 and E2 begin to amplify the viral DNA, BRD4 is displaced from the foci [21]. In order to test whether this is also the case in U2OS cells, we performed an immunofluorescence assay. Six days after transfection with 2 μg of wt HPV11 minicircle genomes, the cells were fixed and immunostained for HPV11 E2 and BRD4 proteins. As shown in Fig. 1, the individual U2OS cells contained different number of HPV11 E2 foci (Fig. 1 HPV11 E2 column). We detected up to ten HPV11 E2 foci, with an average value of four foci per cell nucleus. The HPV replication foci also had a different size. The small HPV11 E2 foci co-localized with the cellular BRD4 protein (Fig. 1a), and were reminiscent of the small early HPV replication foci that were described in human keratinocytes by Sakakibara and others [21]. Large HPV11 E2 foci were also visible in U2OS cells at the same time-point, and the cellular BRD4 protein was absent from these foci (Fig. 1b). Overall, this indicates the involvement of BRD4 in the early steps of replication foci formation in U2OS cells, similar to what has been observed in human keratinocytes [21].Fig. 1

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