High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia mutated (ATM) and the ataxia telangiectasia and Rad3-related (ATR) DNA damage repair pathways for viral genome amplification. HPVs activate these pathways through the immune regulator STAT-5. For the ATR pathway, STAT-5 increases expression of the topoisomerase IIβ-binding protein 1 (TopBP1), a scaffold protein that binds ATR and recruits it to sites of DNA damage. TopBP1 also acts as a transcriptional regulator and we investigated how this activity influenced the HPV life cycle. We determined that TopBP1 levels are increased in cervical intraepithelial neoplasias as well as cervical carcinomas, consistent with studies in HPV-positive cell lines. Suppression of TopBP1 by shRNAs impairs HPV genome amplification and activation of the ATR pathway but does not affect the total levels of ATR and CHK1. In contrast, knockdown reduces the expression of other DNA damage factors such as RAD51 and Mre11 but not BRCA2 or NBS1. Interestingly, TopBP1 positively regulates the expression of E2F1, a TopBP1 binding partner, and p73, in HPV positive cells in contrast to effects in other cell types. TopBP1 transcriptional activity is regulated by AKT and treatment with AKT inhibitors suppresses expression of E2F1 and p73 without interfering with ATR signaling. Importantly, the levels of p73 are elevated in HPV-positive cells and knockdown impairs HPV genome amplification. This demonstrates that p73, like p63 and p53, is an important regulator of the HPV life cycle that is controlled by the transcriptional activating properties of the multifunctional TopBP1 protein.

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.

High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells. IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication.

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.