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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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Related in: MedlinePlus

Cellular replication factors colocalize with the HPV 18 E1 in HeLa cells.Cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) together with the following proteins are shown: HPV18 E2, PCNA, RPA, polymerase α/primase, and Daxx (Alexa Fluor 488, first column). Merged images are presented in the third column and DAPI stained nuclei in the fourth column.
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ppat-1000397-g003: Cellular replication factors colocalize with the HPV 18 E1 in HeLa cells.Cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) together with the following proteins are shown: HPV18 E2, PCNA, RPA, polymerase α/primase, and Daxx (Alexa Fluor 488, first column). Merged images are presented in the third column and DAPI stained nuclei in the fourth column.

Mentions: To further elaborate on the potential functionality of these detected foci, we performed co-immunostaining for E1 and E2, PCNA, the RPA p70 subunit or the polymerase/α-primase (polα) in HeLa cells that were transfected with the HPV18 E1 and E2 expression vectors (Figure 3). We detected the same pattern of co-localization of E1 and the other replication proteins as earlier, which once more demonstrates that these foci are active DNA replication initiation centers of the integrated HPV. This is supported by the fact that the E2 protein is not required for the unwinding or the helicase activity of E1 during DNA replication elongation and, likewise, the fact that polα is not required for DNA repair processes. The percentage of the E1/E2 positive cells with the characteristic pattern of viral DNA replication was estimated to be 5% to 10%, which represents a fairly large fraction of cells in S-phase within the transfected cell population. In the case of the HPV18 replication proteins, we also detected some satellite foci in addition to the main ones in both HeLa cells (Figure 3) and SiHa cells (data not shown). The relative amount of the cells with satellite foci increased over time from 26% at 12 h post-transfection to 74% at 48 h, as evaluated by two independent experiments in the HeLa cells. Co-localization of HPV18 E2, PCNA, the RPA p70 subunit, and polα with HPV E1 indicates that these additional foci may represent the extrachromosomal fraction of the replicating, HPV origin containing, plasmids, which originated from the integrated HPV locus due to its over-amplification. These molecules were clearly detected by the two-dimensional gel-electrophoresis analyses (Figure 1E).


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)

Cellular replication factors colocalize with the HPV 18 E1 in HeLa cells.Cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) together with the following proteins are shown: HPV18 E2, PCNA, RPA, polymerase α/primase, and Daxx (Alexa Fluor 488, first column). Merged images are presented in the third column and DAPI stained nuclei in the fourth column.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666264&req=5

ppat-1000397-g003: Cellular replication factors colocalize with the HPV 18 E1 in HeLa cells.Cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) together with the following proteins are shown: HPV18 E2, PCNA, RPA, polymerase α/primase, and Daxx (Alexa Fluor 488, first column). Merged images are presented in the third column and DAPI stained nuclei in the fourth column.
Mentions: To further elaborate on the potential functionality of these detected foci, we performed co-immunostaining for E1 and E2, PCNA, the RPA p70 subunit or the polymerase/α-primase (polα) in HeLa cells that were transfected with the HPV18 E1 and E2 expression vectors (Figure 3). We detected the same pattern of co-localization of E1 and the other replication proteins as earlier, which once more demonstrates that these foci are active DNA replication initiation centers of the integrated HPV. This is supported by the fact that the E2 protein is not required for the unwinding or the helicase activity of E1 during DNA replication elongation and, likewise, the fact that polα is not required for DNA repair processes. The percentage of the E1/E2 positive cells with the characteristic pattern of viral DNA replication was estimated to be 5% to 10%, which represents a fairly large fraction of cells in S-phase within the transfected cell population. In the case of the HPV18 replication proteins, we also detected some satellite foci in addition to the main ones in both HeLa cells (Figure 3) and SiHa cells (data not shown). The relative amount of the cells with satellite foci increased over time from 26% at 12 h post-transfection to 74% at 48 h, as evaluated by two independent experiments in the HeLa cells. Co-localization of HPV18 E2, PCNA, the RPA p70 subunit, and polα with HPV E1 indicates that these additional foci may represent the extrachromosomal fraction of the replicating, HPV origin containing, plasmids, which originated from the integrated HPV locus due to its over-amplification. These molecules were clearly detected by the two-dimensional gel-electrophoresis analyses (Figure 1E).

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