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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 DAXX protein. a and b 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 DAXX (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 FISH analysis of U2OS cells transfected with HPV18 genome. U2OS cells were transfected with wt HPV18 minicircle genomes and grown on microscope slides. Six days after transfection cells were fixed and combined immunofluorescence and FISH analysis was performed. DAXX signal is visible in red and HPV18 DNA in green. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. On the right panels, the intensity profiles are shown
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Fig2: The localization of HPV replication foci in U2OS cells in relation to the DAXX protein. a and b 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 DAXX (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 FISH analysis of U2OS cells transfected with HPV18 genome. U2OS cells were transfected with wt HPV18 minicircle genomes and grown on microscope slides. Six days after transfection cells were fixed and combined immunofluorescence and FISH analysis was performed. DAXX signal is visible in red and HPV18 DNA in green. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. On the right panels, the intensity profiles are shown

Mentions: The main aim of this work was to analyze the localization of HPV replication centers in relation to components of ND10 in U2OS cells. Many DNA viruses replicate their genomes in close proximity to ND10, and several reports link multiple stages of HPV infection with these nuclear sites [4, 5, 22, 23]. However, the role of ND10 in papillomavirus infection is still controversial. In order to analyze the link between HPV replication and ND10 in U2OS cells, we first analyzed the localization of the HPV E2 protein and cellular DAXX protein, a constitutive component of ND10. For this purpose, U2OS cells were transfected with 2 μg of wt HPV11 minicircle genomes, fixed six days later and immunostained for HPV11 E2 and cellular DAXX proteins. On average, seven DAXX-containing foci per cell nucleus were observed (Fig. 2 DAXX column), and 34 % of the E2 foci overlapped partially with DAXX-containing foci. Representative images of cells with different number and size of HPV11 E2 foci and DAXX staining are shown in Fig. 2a and b. A similar analysis with HPV18 replication centers failed due to the fact that we were unable to find mouse monoclonal antibodies against the DAXX protein suitable for immunofluorescence analysis. Therefore, we performed immunofluorescence analysis coupled with FISH detection of HPV18 DNA. U2OS cells were transfected with 5 μg of wt HPV18 minicircle genomes and fixed six days later. The cells were immunostained for DAXX protein and HPV DNA was detected with an HPV18 specific probe (Fig. 2c). We observed at least partial co-localization of DAXX and HPV DNA in 22 % of the HPV18 DNA foci in U2OS cells.Fig. 2


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 DAXX protein. a and b 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 DAXX (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 FISH analysis of U2OS cells transfected with HPV18 genome. U2OS cells were transfected with wt HPV18 minicircle genomes and grown on microscope slides. Six days after transfection cells were fixed and combined immunofluorescence and FISH analysis was performed. DAXX signal is visible in red and HPV18 DNA in green. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. 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

Fig2: The localization of HPV replication foci in U2OS cells in relation to the DAXX protein. a and b 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 DAXX (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 FISH analysis of U2OS cells transfected with HPV18 genome. U2OS cells were transfected with wt HPV18 minicircle genomes and grown on microscope slides. Six days after transfection cells were fixed and combined immunofluorescence and FISH analysis was performed. DAXX signal is visible in red and HPV18 DNA in green. Cell nuclei were detected by DAPI. Analysis was carried out at least three times starting from cell transfections. On the right panels, the intensity profiles are shown
Mentions: The main aim of this work was to analyze the localization of HPV replication centers in relation to components of ND10 in U2OS cells. Many DNA viruses replicate their genomes in close proximity to ND10, and several reports link multiple stages of HPV infection with these nuclear sites [4, 5, 22, 23]. However, the role of ND10 in papillomavirus infection is still controversial. In order to analyze the link between HPV replication and ND10 in U2OS cells, we first analyzed the localization of the HPV E2 protein and cellular DAXX protein, a constitutive component of ND10. For this purpose, U2OS cells were transfected with 2 μg of wt HPV11 minicircle genomes, fixed six days later and immunostained for HPV11 E2 and cellular DAXX proteins. On average, seven DAXX-containing foci per cell nucleus were observed (Fig. 2 DAXX column), and 34 % of the E2 foci overlapped partially with DAXX-containing foci. Representative images of cells with different number and size of HPV11 E2 foci and DAXX staining are shown in Fig. 2a and b. A similar analysis with HPV18 replication centers failed due to the fact that we were unable to find mouse monoclonal antibodies against the DAXX protein suitable for immunofluorescence analysis. Therefore, we performed immunofluorescence analysis coupled with FISH detection of HPV18 DNA. U2OS cells were transfected with 5 μg of wt HPV18 minicircle genomes and fixed six days later. The cells were immunostained for DAXX protein and HPV DNA was detected with an HPV18 specific probe (Fig. 2c). We observed at least partial co-localization of DAXX and HPV DNA in 22 % of the HPV18 DNA foci in U2OS cells.Fig. 2

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