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Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

Bottom Line: These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH.We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV.It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
In HPV-related cancers, the "high-risk" human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the "onion skin"-type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone gammaH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV-replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV-associated cancers. It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

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DNA replication of the integrated HPV takes place at specific nuclear foci.HeLa and SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Cells were analyzed 20 hrs post-transfection. (A) Co-immunostaining of E1 (Alexa Fluor 568, first column) and BrdU (FITC, second column). Cells were pulse-labeled with BrdU for 2 hrs prior to IF analysis. In the third column, the localizations of E1 and BrdU in the same cells are presented as a merged image. (B) Combined immunofluorescence and FISH analysis to detect the integrated HPV DNA (Alexa Fluor 488, first column) and the HPV E1 protein (Alexa Fluor 568, second column) in SiHa and HeLa cells. Localizations of the E1 and the integrated HPV DNA are also presented in the third column as a merged image. DNA was counterstained with DAPI (fourth column).
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ppat-1000397-g002: DNA replication of the integrated HPV takes place at specific nuclear foci.HeLa and SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Cells were analyzed 20 hrs post-transfection. (A) Co-immunostaining of E1 (Alexa Fluor 568, first column) and BrdU (FITC, second column). Cells were pulse-labeled with BrdU for 2 hrs prior to IF analysis. In the third column, the localizations of E1 and BrdU in the same cells are presented as a merged image. (B) Combined immunofluorescence and FISH analysis to detect the integrated HPV DNA (Alexa Fluor 488, first column) and the HPV E1 protein (Alexa Fluor 568, second column) in SiHa and HeLa cells. Localizations of the E1 and the integrated HPV DNA are also presented in the third column as a merged image. DNA was counterstained with DAPI (fourth column).

Mentions: DNA replication of eukaryotic cells occurs within defined sites throughout the nucleus, as identified by co-localization of replication factors and nascent bromodeoxyuridine (BrdU) labeled DNA into distinct foci [42]–[44]. It has also been demonstrated that papillomaviruses, which are similar to many other DNA viruses, replicate their genome at specific nuclear extrachromosomal foci in infected cells [45]. To identify the replication sites of integrated HPV, we transfected SiHa cells with HPV16 E1 and E2 expression vectors and HeLa cells with HPV18 E1 and E2 expression vectors. Twenty hours post-transfection, the cells were labeled with BrdU for 2 hours, followed by double immunostaining for BrdU and HA-tagged HPV E1 protein. The results show that BrdU was incorporated throughout the nucleus in the cells without the E1 protein. However, in cells that were positive for E1 and E2, the BrdU signal was mostly co-localized within the E1 foci (Figure 2A). In such cases, we always identified 2 foci in the SiHa cells and 3 foci in the HeLa cells, although they were heterogeneous in size and there was a tendency for satellite foci later in the time course (Figure 2A). These foci likely represent the integrated HPV sites that are capable of active replication in these cell lines. The SiHa cells contain two chromosome 13's that carry the single HPV16 integration site [46]–[48], which suggests that both copies of the HPV16 are active for replication. In HeLa cells, at least five HPV18 integration sites have been mapped. Three of them are located on normal chromosomes 8 at 8q24 and two on derivative chromosomes, which have been shown to contain material from 8q24 [49]. We conclude that there are three replication competent loci of the HPV18 in HeLa cells. BrdU incorporation within the E1 foci indicates that the replication of the integrated HPV origin can be visualized by E1 immunofluorescence analysis and that such compartmentalization of E1 to these foci occurs only during S-phase of the cell cycle. To confirm that the visualized foci represent the HPV replication centers, the immunostaining for E1 was combined with FISH analysis for the integrated HPV-specific DNA (Figure 2B). The amplified DNA of HPV16 (in SiHa cells) and HPV18 (in HeLa cells) are detected at the same foci as the E1 protein (Figure 2B). These data again suggest that the E1 foci are bona fide amplification sites of the integrated HPV.


Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

DNA replication of the integrated HPV takes place at specific nuclear foci.HeLa and SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Cells were analyzed 20 hrs post-transfection. (A) Co-immunostaining of E1 (Alexa Fluor 568, first column) and BrdU (FITC, second column). Cells were pulse-labeled with BrdU for 2 hrs prior to IF analysis. In the third column, the localizations of E1 and BrdU in the same cells are presented as a merged image. (B) Combined immunofluorescence and FISH analysis to detect the integrated HPV DNA (Alexa Fluor 488, first column) and the HPV E1 protein (Alexa Fluor 568, second column) in SiHa and HeLa cells. Localizations of the E1 and the integrated HPV DNA are also presented in the third column as a merged image. DNA was counterstained with DAPI (fourth column).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666264&req=5

ppat-1000397-g002: DNA replication of the integrated HPV takes place at specific nuclear foci.HeLa and SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Cells were analyzed 20 hrs post-transfection. (A) Co-immunostaining of E1 (Alexa Fluor 568, first column) and BrdU (FITC, second column). Cells were pulse-labeled with BrdU for 2 hrs prior to IF analysis. In the third column, the localizations of E1 and BrdU in the same cells are presented as a merged image. (B) Combined immunofluorescence and FISH analysis to detect the integrated HPV DNA (Alexa Fluor 488, first column) and the HPV E1 protein (Alexa Fluor 568, second column) in SiHa and HeLa cells. Localizations of the E1 and the integrated HPV DNA are also presented in the third column as a merged image. DNA was counterstained with DAPI (fourth column).
Mentions: DNA replication of eukaryotic cells occurs within defined sites throughout the nucleus, as identified by co-localization of replication factors and nascent bromodeoxyuridine (BrdU) labeled DNA into distinct foci [42]–[44]. It has also been demonstrated that papillomaviruses, which are similar to many other DNA viruses, replicate their genome at specific nuclear extrachromosomal foci in infected cells [45]. To identify the replication sites of integrated HPV, we transfected SiHa cells with HPV16 E1 and E2 expression vectors and HeLa cells with HPV18 E1 and E2 expression vectors. Twenty hours post-transfection, the cells were labeled with BrdU for 2 hours, followed by double immunostaining for BrdU and HA-tagged HPV E1 protein. The results show that BrdU was incorporated throughout the nucleus in the cells without the E1 protein. However, in cells that were positive for E1 and E2, the BrdU signal was mostly co-localized within the E1 foci (Figure 2A). In such cases, we always identified 2 foci in the SiHa cells and 3 foci in the HeLa cells, although they were heterogeneous in size and there was a tendency for satellite foci later in the time course (Figure 2A). These foci likely represent the integrated HPV sites that are capable of active replication in these cell lines. The SiHa cells contain two chromosome 13's that carry the single HPV16 integration site [46]–[48], which suggests that both copies of the HPV16 are active for replication. In HeLa cells, at least five HPV18 integration sites have been mapped. Three of them are located on normal chromosomes 8 at 8q24 and two on derivative chromosomes, which have been shown to contain material from 8q24 [49]. We conclude that there are three replication competent loci of the HPV18 in HeLa cells. BrdU incorporation within the E1 foci indicates that the replication of the integrated HPV origin can be visualized by E1 immunofluorescence analysis and that such compartmentalization of E1 to these foci occurs only during S-phase of the cell cycle. To confirm that the visualized foci represent the HPV replication centers, the immunostaining for E1 was combined with FISH analysis for the integrated HPV-specific DNA (Figure 2B). The amplified DNA of HPV16 (in SiHa cells) and HPV18 (in HeLa cells) are detected at the same foci as the E1 protein (Figure 2B). These data again suggest that the E1 foci are bona fide amplification sites of the integrated HPV.

Bottom Line: These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH.We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV.It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
In HPV-related cancers, the "high-risk" human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the "onion skin"-type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone gammaH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV-replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV-associated cancers. It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

Show MeSH
Related in: MedlinePlus