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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: In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.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.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 early gene expression. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 or wt HPV11 genome. 48 h after transfection total RNA was isolated, reverse transcribed and analyzed by qPCR. Each sample was analyzed in triplicate with E6, E7, E1, E2, beta-actin and HPRT1 specific primers. HPV11 and 18 (a) and bACT (b) mRNA levels were calculated relative to HPRT1 mRNA levels. The data represent the average of three independent experiments. Blow-out of HPV18 part from (a) is shown in (c). d The location of four different primer pairs targeted against HPV E6, E7, E1 and E2 coding sequences in HPV18 early region
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Fig5: Down-regulation of DAXX represses HPV early gene expression. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 or wt HPV11 genome. 48 h after transfection total RNA was isolated, reverse transcribed and analyzed by qPCR. Each sample was analyzed in triplicate with E6, E7, E1, E2, beta-actin and HPRT1 specific primers. HPV11 and 18 (a) and bACT (b) mRNA levels were calculated relative to HPRT1 mRNA levels. The data represent the average of three independent experiments. Blow-out of HPV18 part from (a) is shown in (c). d The location of four different primer pairs targeted against HPV E6, E7, E1 and E2 coding sequences in HPV18 early region

Mentions: The HPV genomes are transcriptionally active in U2OS cells and express all the viral early proteins required for initiation and establishment of HPV replication [19, 26]. In order to determine whether the DAXX protein is involved in papillomavirus early promoter regulation, we studied the expression of viral early genes that were transcribed from wt HPV18 and wt HPV11 episomes in U2OS cells. Four different primer pairs were used to measure the activity of HPV early promoters. E6, E7, E1 and E2 primers target the corresponding coding sequences in the viral genome (Fig. 5d). Most of these primers target multiple HPV transcripts in U2OS cells [26]. HPV18 E6 primers detect six differently spliced transcripts from early promoter P102; HPV18 E7 primers detect the same six transcripts, along with an additional three transcripts from promoter P520. HPV18 E1 primers detect a single E1 encoding transcript originating from promoter P102, and HPV18 E2 primers detect transcripts from three different promoters (P102, P811 and P1193). HPV11 primers were targeted to similar positions in the HPV11 genome.Fig. 5


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 early gene expression. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 or wt HPV11 genome. 48 h after transfection total RNA was isolated, reverse transcribed and analyzed by qPCR. Each sample was analyzed in triplicate with E6, E7, E1, E2, beta-actin and HPRT1 specific primers. HPV11 and 18 (a) and bACT (b) mRNA levels were calculated relative to HPRT1 mRNA levels. The data represent the average of three independent experiments. Blow-out of HPV18 part from (a) is shown in (c). d The location of four different primer pairs targeted against HPV E6, E7, E1 and E2 coding sequences in HPV18 early region
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Down-regulation of DAXX represses HPV early gene expression. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 or wt HPV11 genome. 48 h after transfection total RNA was isolated, reverse transcribed and analyzed by qPCR. Each sample was analyzed in triplicate with E6, E7, E1, E2, beta-actin and HPRT1 specific primers. HPV11 and 18 (a) and bACT (b) mRNA levels were calculated relative to HPRT1 mRNA levels. The data represent the average of three independent experiments. Blow-out of HPV18 part from (a) is shown in (c). d The location of four different primer pairs targeted against HPV E6, E7, E1 and E2 coding sequences in HPV18 early region
Mentions: The HPV genomes are transcriptionally active in U2OS cells and express all the viral early proteins required for initiation and establishment of HPV replication [19, 26]. In order to determine whether the DAXX protein is involved in papillomavirus early promoter regulation, we studied the expression of viral early genes that were transcribed from wt HPV18 and wt HPV11 episomes in U2OS cells. Four different primer pairs were used to measure the activity of HPV early promoters. E6, E7, E1 and E2 primers target the corresponding coding sequences in the viral genome (Fig. 5d). Most of these primers target multiple HPV transcripts in U2OS cells [26]. HPV18 E6 primers detect six differently spliced transcripts from early promoter P102; HPV18 E7 primers detect the same six transcripts, along with an additional three transcripts from promoter P520. HPV18 E1 primers detect a single E1 encoding transcript originating from promoter P102, and HPV18 E2 primers detect transcripts from three different promoters (P102, P811 and P1193). HPV11 primers were targeted to similar positions in the HPV11 genome.Fig. 5

Bottom Line: In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.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.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