Interplay between the DNA damage response and the life cycle of DNA tumor viruses.

Approximately 20 % of human cancers are associated with virus infection. DNA tumor viruses can induce tumor formation in host cells by disrupting the cell's DNA replication and repair mechanisms. Specifically, these viruses interfere with the host cell's DNA damage response (DDR), which is a complex network of signaling pathways that is essential for maintaining the integrity of the genome. DNA tumor viruses can disrupt these pathways by expressing oncoproteins that mimic or inhibit various DDR components, thereby promoting genomic instability and tumorigenesis. Recent studies have highlighted the molecular mechanisms by which DNA tumor viruses interact with DDR components, as well as the ways in which these interactions contribute to viral replication and tumorigenesis. Understanding the interplay between DNA tumor viruses and the DDR pathway is critical for developing effective strategies to prevent and treat virally associated cancers. In this review, we discuss the current state of knowledge regarding the mechanisms by which human papillomavirus (HPV), merkel cell polyomavirus (MCPyV), Kaposi's sarcoma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV) interfere with DDR pathways to facilitate their respective life cycles, and the consequences of such interference on genomic stability and cancer development.

The SETD2 Methyltransferase Supports Productive HPV31 Replication through the LEDGF/CtIP/Rad51 Pathway.

The human papillomavirus (HPV) life cycle takes place in the stratified epithelium, with the productive phase being activated by epithelial differentiation. The HPV genome is histone-associated, and the life cycle is epigenetically regulated, in part, by histone tail modifications that facilitate the recruitment of DNA repair factors that are required for viral replication. We previously showed that the SETD2 methyltransferase facilitates the productive replication of HPV31 through the trimethylation of H3K36 on viral chromatin. SETD2 regulates numerous cellular processes, including DNA repair via homologous recombination (HR) and alternative splicing, through the recruitment of various effectors to histone H3 lysine 36 trimethylation (H3K36me3). We previously demonstrated that the HR factor Rad51 is recruited to HPV31 genomes and is required for productive replication; however, the mechanism of Rad51 recruitment has not been defined. SET domain containing 2 (SETD2) promotes the HR repair of double-strand breaks (DSBs) in actively transcribed genes through the recruitment of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) to lens epithelium-derived growth factor (LEDGF)-bound H3K36me3, which promotes DNA end resection and thereby allows for the recruitment of Rad51 to damaged sites. In this study, we found that reducing H3K36me3 through the depletion of SETD2 or the overexpression of an H3.3K36M mutant leads to an increase in γH2AX, which is a marker of damage, on viral DNA upon epithelial differentiation. This is coincident with decreased Rad51 binding. Additionally, LEDGF and CtIP are bound to HPV DNA in a SETD2-dependent and H3K36me3-dependent manner, and they are required for productive replication. Furthermore, CtIP depletion increases DNA damage on viral DNA and blocks Rad51 recruitment upon differentiation. Overall, these studies indicate that H3K36me3 enrichment on transcriptionally active viral genes promotes the rapid repair of viral DNA upon differentiation through the LEDGF-CtIP-Rad51 axis. IMPORTANCE The productive phase of the HPV life cycle is restricted to the differentiating cells of the stratified epithelium. The HPV genome is histone-associated and subject to epigenetic regulation, though the manner in which epigenetic modifications contribute to productive replication is largely undefined. In this study, we demonstrate that SETD2-mediated H3K36me3 on HPV31 chromatin promotes productive replication through the repair of damaged DNA. We show that SETD2 facilitates the recruitment of the homologous recombination repair factors CtIP and Rad51 to viral DNA through LEDGF binding to H3K36me3. CtIP is recruited to damaged viral DNA upon differentiation, and, in turn, recruits Rad51. This likely occurs through the end resection of double-strand breaks. SETD2 trimethylates H3K36me3 during transcription, and active transcription is necessary for Rad51 recruitment to viral DNA. We propose that the enrichment of SETD2-mediated H3K36me3 on transcriptionally active viral genes upon differentiation facilitates the repair of damaged viral DNA during the productive phase of the viral life cycle.

For Better or Worse: Modulation of the Host DNA Damage Response by Human Papillomavirus.

High-risk human papillomaviruses (HPVs) are associated with several human cancers. HPVs are small, DNA viruses that rely on host cell machinery for viral replication. The HPV life cycle takes place in the stratified epithelium, which is composed of different cell states, including terminally differentiating cells that are no longer active in the cell cycle. HPVs have evolved mechanisms to persist and replicate in the stratified epithelium by hijacking and modulating cellular pathways, including the DNA damage response (DDR). HPVs activate and exploit DDR pathways to promote viral replication, which in turn increases the susceptibility of the host cell to genomic instability and carcinogenesis. Here, we review recent advances in our understanding of the regulation of the host cell DDR by high-risk HPVs during the viral life cycle and discuss the potential cellular consequences of modulating DDR pathways.

Regulation of the Innate Immune Response during the Human Papillomavirus Life Cycle.

High-risk human papillomaviruses (HR HPVs) are associated with multiple human cancers and comprise 5% of the human cancer burden. Although most infections are transient, persistent infections are a major risk factor for cancer development. The life cycle of HPV is intimately linked to epithelial differentiation. HPVs establish infection at a low copy number in the proliferating basal keratinocytes of the stratified epithelium. In contrast, the productive phase of the viral life cycle is activated upon epithelial differentiation, resulting in viral genome amplification, high levels of late gene expression, and the assembly of virions that are shed from the epithelial surface. Avoiding activation of an innate immune response during the course of infection plays a key role in promoting viral persistence as well as completion of the viral life cycle in differentiating epithelial cells. This review highlights the recent advances in our understanding of how HPVs manipulate the host cell environment, often in a type-specific manner, to suppress activation of an innate immune response to establish conditions supportive of viral replication.

Epigenetic Regulation of the Human Papillomavirus Life Cycle.

Persistent infection with certain types of human papillomaviruses (HPVs), termed high risk, presents a public health burden due to their association with multiple human cancers, including cervical cancer and an increasing number of head and neck cancers. Despite the development of prophylactic vaccines, the incidence of HPV-associated cancers remains high. In addition, no vaccine has yet been licensed for therapeutic use against pre-existing HPV infections and HPV-associated diseases. Although persistent HPV infection is the major risk factor for cancer development, additional genetic and epigenetic alterations are required for progression to the malignant phenotype. Unlike genetic mutations, the reversibility of epigenetic modifications makes epigenetic regulators ideal therapeutic targets for cancer therapy. This review article will highlight the recent advances in the understanding of epigenetic modifications associated with HPV infections, with a particular focus on the role of these epigenetic changes during different stages of the HPV life cycle that are closely associated with activation of DNA damage response pathways.

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response.

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Impact of Replication Stress in Human Papillomavirus Pathogenesis.

The inactivation of critical cell cycle checkpoints by the human papillomavirus (HPV) oncoprotein E7 results in replication stress (RS) that leads to genomic instability in premalignant lesions. Intriguingly, RS tolerance is achieved through several mechanisms, enabling HPV to exploit the cellular RS response for viral replication and to facilitate viral persistence in the presence of DNA damage. As such, inhibitors of the RS response pathway may provide a novel approach to target HPV-associated lesions and cancers.

SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle.

The life cycle of HPV is tied to the differentiation status of its host cell, with productive replication, late gene expression and virion production restricted to the uppermost layers of the stratified epithelium. HPV DNA is histone-associated, exhibiting a chromatin structure similar to that of the host chromosome. Although HPV chromatin is subject to histone post-translational modifications, how the viral life cycle is epigenetically regulated is not well understood. SETD2 is a histone methyltransferase that places the trimethyl mark on H3K36 (H3K36me3), a mark of active transcription. Here, we define a role for SETD2 and H3K36me3 in the viral life cycle. We have found that HPV positive cells exhibit increased levels of SETD2, with SETD2 depletion leading to defects in productive viral replication and splicing of late viral RNAs. Reducing H3K36me3 by overexpression of KDM4A, an H3K36me3 demethylase, or an H3.3K36M transgene also blocks productive viral replication, indicating a significant role for this histone modification in facilitating viral processes. H3K36me3 is enriched on the 3' end of the early region of the high-risk HPV31 genome in a SETD2-dependent manner, suggesting that SETD2 may regulate the viral life cycle through the recruitment of H3K36me3 readers to viral DNA. Intriguingly, we have found that activation of the ATM DNA damage kinase, which is required for productive viral replication, is necessary for the maintenance of H3K36me3 on viral chromatin and for processing of late viral RNAs. Additionally, we have found that the HPV31 E7 protein maintains the increased SETD2 levels in infected cells through an extension of protein half-life. Collectively, our findings highlight the importance of epigenetic modifications in driving the viral life cycle and identify a novel role for E7 as well as the DNA damage response in the regulation of viral processes through epigenetic modifications.

Air-Liquid Interface System To Understand Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) infects epithelial cells and is associated with epithelial malignancies. Although EBV reactivation is induced by epithelial differentiation, the available methods for differentiation are not widely used. In a recent study, Caves et al. (mSphere 3:e00152-18, 2018, https://doi.org/10.1128/mSphere.00152-18) explored the use of a new transwell-based air-liquid interface (ALI) system to differentiate EBV-infected nasopharyngeal carcinoma cells. They found that cells cultured in the ALI system expressed markers of differentiation and supported complete EBV reactivation. This system offers an easy method for differentiation that could be widely adopted. This system could be extended to other epithelial cell types.

The Rb binding domain of HPV31 E7 is required to maintain high levels of DNA repair factors in infected cells.

Human papillomaviruses (HPV) exhibit constitutive activation of ATM and ATR DNA damage response (DDR) pathways, which are required for productive viral replication. Expression of HPV31 E7 alone is sufficient to activate the DDR through an unknown mechanism. Here, we demonstrate that the E7 Rb binding domain is required to increase levels of many DDR proteins, including ATM, Chk2, Chk1, the MRN components MRE11, Rad50, and NBS1, as well as the homologous recombination repair proteins BRCA1 and Rad51. Interestingly, we have found that the increase in these DNA repair proteins does not occur solely at the level of transcription, but that E7 broadly increases the half-life of these DDR factors, a phenotype that is lost in the E7 Rb binding mutant. These data suggest that HPV-31 upregulates DNA repair factors necessary for replication by increasing protein half-life in a manner requiring the E7 Rb binding domain.

Modulation of the DNA damage response during the life cycle of human papillomaviruses.

Human papillomavirus (HPV) is the most common sexually transmitted viral infection. Infection with certain types of HPV pose a major public health risk as these types are associated with multiple human cancers, including cervical cancer, other anogenital malignancies and an increasing number of head and neck cancers. The HPV life cycle is closely tied to host cell differentiation with late viral events such as structural gene expression and viral genome amplification taking place in the upper layers of the stratified epithelium. The DNA damage response (DDR) is an elaborate signaling network of proteins that regulate the fidelity of replication by detecting, signaling and repairing DNA lesions. ATM and ATR are two kinases that are major regulators of DNA damage detection and repair. A multitude of studies indicate that activation of the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) pathways are critical for HPV to productively replicate. This review outlines how HPV interfaces with the ATM- and ATR-dependent DNA damage responses throughout the viral life cycle to create an environment supportive of viral replication and how activation of these pathways could impact genomic stability.

HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes.

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

UNLABELLED: High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE: Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle.

LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1.

UNLABELLED: As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV OriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency. IMPORTANCE: Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by a ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to antiviral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to antiviral therapies.

Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

UNLABELLED: Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE: The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.

Human papillomaviruses recruit cellular DNA repair and homologous recombination factors to viral replication centers.

Human papillomaviruses (HPV) activate the ataxia telangiectasia mutated (ATM)-dependent DNA damage response to induce viral genome amplification upon epithelial differentiation. Our studies show that along with members of the ATM pathway, HPV proteins also localize factors involved in homologous DNA recombination to distinct nuclear foci that contain HPV genomes and cellular replication factors. These studies indicate that HPV activates the ATM pathway to recruit repair factors to viral genomes and allow for efficient replication.

Nuclear accumulation of the papillomavirus E1 helicase blocks S-phase progression and triggers an ATM-dependent DNA damage response.

Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1

The G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK-cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence ((43)RRLL(46)) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events.