Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

Orav, Marit; Gagnon, David; Archambault, Jacques

doi:10.1128/mbio.00152-19

Cited by 10 publications

(16 citation statements)

References 64 publications

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“…In addition, TOP2b or other topoisomerases may play a critical role in helping resolve concatemeric viral genomes resulting from amplification. Amplification has been reported to occur through several potential mechanisms, including a rolling circle type process or a novel unidirectional replication scheme involving multiple initiation sites (21,22). It is also possible that amplification occurs though bidirectional theta structures.…”

Section: Discussionmentioning

confidence: 99%

“…Keratinocytes were induced to differentiate through a calciummediated switch (3). Briefly, keratinocytes were plated at 5 million cells per 10-cm dish in M154 keratinocyte medium (Thermo Fisher) containing 0.07 mM Ca 21 and human keratinocyte growth serum (HKGS; Thermo Fisher). The next day, the medium was changed to 0.03 mM Ca 21 in M154 containing HKGS.…”

Section: Methodsmentioning

confidence: 99%

“…The next day, the medium was changed to 0.03 mM Ca 21 in M154 containing HKGS. After 24 h, the medium was changed once again to M154 containing 1.5 mM Ca 21 in the absence of HKGS. These cells were then incubated in high-calcium medium for 72 h to allow for proper differentiation.…”

Section: Methodsmentioning

confidence: 99%

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Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

Kaminski

Hong

Kono

et al. 2021

mBio

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Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2β. Upon DNA cleavage, a covalent intermediate DNA-TOP2β (TOP2βcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2βcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2β proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2β and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2β with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2β also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2β. This study identifies TOP2β as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication. IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2β levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2β reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2β through DNA break formation.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

Kaminski

Hong

Kono

et al. 2021

mBio

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show abstract

“…In addition, TOP2 or other topoisomerases may play a critical role in helping resolve concatemeric viral genomes resulting from amplification. Amplification has been reported to occur through several potential mechanisms including a rolling circle type process or a novel unidirectional replication scheme involving multiple initiation sites (22,23). It also is possible that amplification occurs though bi-directional theta structures.…”

mentioning

confidence: 99%

Topoisomerase 2β induces DNA breaks to regulate human papillomavirus replication

Kaminski¹,

Hong²,

Kono³

et al. 2021

Preprint

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Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2β is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2β. Upon DNA cleavage a covalent intermediate DNA-TOP2β (TOP2βcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2βcc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2β proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2β and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2β with shRNAs reduced DNA breaks by over 50% as determined through COMET assays. Furthermore this correlated with substantially reduced formation of repair foci such as γH2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2β also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2β. This study identifies TOP2β as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

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“…The question of whether the maintenance replication of the HPV genome is under the control of the cellular origin licensing machinery and restricted to one replication per cell cycle, or whether replication is statistically completed once per cell cycle, has remained obscure, since both options have been proposed (8,9). Second, it has been shown that a novel mode of replication is initiated already during the transient replication of the viral genome, as determined by two-dimensional (2D) gel electrophoresis analysis of replication intermediates (10,11). The exact nature of this novel mode of replication has remained enigmatic, but it has been suggested to involve cellular recombination-dependent replication machinery (10).…”

mentioning

confidence: 99%

Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

Piirsoo

Kala

Sankovski

et al. 2020

J Virol

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The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication – initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins, and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function. IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes, maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases – replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

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Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

Cited by 10 publications

References 64 publications

Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

Topoisomerase 2β induces DNA breaks to regulate human papillomavirus replication

Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

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