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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

The localization of HPV replication foci in U2OS cells in relation to the PML protein. Immunofluorescene analysis of U2OS cells transfected with HPV11 and HPV18E8- genomes. a and b 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 PML (red) proteins. 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. c, d, e and f U2OS cells were transfected with HPV18E8- minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV18 E1 (green) and PML (red) proteins. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. c, d, e and f represent cells with different number and size of HPV18 E1 foci. On the right panels, the intensity profiles are shown
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Fig3: The localization of HPV replication foci in U2OS cells in relation to the PML protein. Immunofluorescene analysis of U2OS cells transfected with HPV11 and HPV18E8- genomes. a and b 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 PML (red) proteins. 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. c, d, e and f U2OS cells were transfected with HPV18E8- minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV18 E1 (green) and PML (red) proteins. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. c, d, e and f represent cells with different number and size of HPV18 E1 foci. On the right panels, the intensity profiles are shown

Mentions: In order to confirm the partial co-localization of HPV replication centers and ND10, immunofluorescence analysis was performed with another component of ND10, the PML protein. First U2OS cells were transfected with 2 μg of wt HPV11 minicircle genomes and immunostained for HPV11 E2 and cellular PML proteins. The PML signal was observed in a punctate pattern in the cell nucleus, with an average of 14 PML structures per nucleus (Fig. 3a, b; PML column). 44 % of the E2 foci overlapped partially with PML-containing foci. In many cases, HPV replication foci and ND10 localized side by side. Representative images of cells with different number and size of HPV11 E2 foci and PML staining are shown in Fig. 3a and b. A similar analysis was performed with HPV18; however, in this case the immunofluorescence analysis was carried out with HPV18E8- genome. The HPV18E8- mutant replicates at a higher level than the wt HPV18 genome [24, 25], resulting in a higher and more easily detectable HPV18 E1 protein level in the transfected cells. In the case of the HPV18E8- genome, we detected up to 24 E1 foci per cell nucleus, with an average of 10 foci per cell. Examples of typical cells with different sizes of E1 foci are shown in Fig. 3c, d, e and f. 42 % of the E1 foci overlapped, at least partially, with PML signal in U2OS cells six days after transfection. However, in most of the cells, there was no perfect co-localization of HPV18 E1 protein and PML signals; rather, a portion of the E1 foci overlapped with PML partially. In a few cells, we were able to detect very small E1 foci surrounded by the PML protein, as shown in Fig. 3c.Fig. 3


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

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

The localization of HPV replication foci in U2OS cells in relation to the PML protein. Immunofluorescene analysis of U2OS cells transfected with HPV11 and HPV18E8- genomes. a and b 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 PML (red) proteins. 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. c, d, e and f U2OS cells were transfected with HPV18E8- minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV18 E1 (green) and PML (red) proteins. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. c, d, e and f represent cells with different number and size of HPV18 E1 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

Fig3: The localization of HPV replication foci in U2OS cells in relation to the PML protein. Immunofluorescene analysis of U2OS cells transfected with HPV11 and HPV18E8- genomes. a and b 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 PML (red) proteins. 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. c, d, e and f U2OS cells were transfected with HPV18E8- minicircle genomes and grown on coverslips. Six days after transfection cells were fixed and immunostained with antibodies for HPV18 E1 (green) and PML (red) proteins. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. c, d, e and f represent cells with different number and size of HPV18 E1 foci. On the right panels, the intensity profiles are shown
Mentions: In order to confirm the partial co-localization of HPV replication centers and ND10, immunofluorescence analysis was performed with another component of ND10, the PML protein. First U2OS cells were transfected with 2 μg of wt HPV11 minicircle genomes and immunostained for HPV11 E2 and cellular PML proteins. The PML signal was observed in a punctate pattern in the cell nucleus, with an average of 14 PML structures per nucleus (Fig. 3a, b; PML column). 44 % of the E2 foci overlapped partially with PML-containing foci. In many cases, HPV replication foci and ND10 localized side by side. Representative images of cells with different number and size of HPV11 E2 foci and PML staining are shown in Fig. 3a and b. A similar analysis was performed with HPV18; however, in this case the immunofluorescence analysis was carried out with HPV18E8- genome. The HPV18E8- mutant replicates at a higher level than the wt HPV18 genome [24, 25], resulting in a higher and more easily detectable HPV18 E1 protein level in the transfected cells. In the case of the HPV18E8- genome, we detected up to 24 E1 foci per cell nucleus, with an average of 10 foci per cell. Examples of typical cells with different sizes of E1 foci are shown in Fig. 3c, d, e and f. 42 % of the E1 foci overlapped, at least partially, with PML signal in U2OS cells six days after transfection. However, in most of the cells, there was no perfect co-localization of HPV18 E1 protein and PML signals; rather, a portion of the E1 foci overlapped with PML partially. In a few cells, we were able to detect very small E1 foci surrounded by the PML protein, as shown in Fig. 3c.Fig. 3

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