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Tumour Virus Res
Tumour Virus Res
Tumour Virus Research
2666-6790
Elsevier

S2666-6790(24)00015-6
10.1016/j.tvr.2024.200291
200291
Review Article
A new role for human papillomavirus 16 E2: Mitotic activation of the DNA damage response to promote viral genome segregation
Prabhakar Apurva T. tadimaripraba@vcu.edu
a
Morgan Iain M. immorgan@vcu.edu
ab⁎
a Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, VA, 23298, USA
b VCU Massey Cancer Center, Richmond, VA, 23298, USA
⁎ Corresponding author. Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, VA, 23298, USA. immorgan@vcu.edu
07 9 2024
12 2024
07 9 2024
18 20029128 6 2024
3 9 2024
5 9 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Human papillomaviruses (HPV) are causative agents in around 5% of all human cancers. To identify and develop new targeted HPV therapeutics we must enhance our understanding of the viral life cycle and how it interacts with the host. The HPV E2 protein dimerizes and binds to 12bp target sequences in the viral genome and segregates the viral genome during mitosis. In this function, E2 binds to the viral genome and the host chromatin simultaneously, ensuring viral genomes reside in daughter nuclei following cell division. We have demonstrated that a mitotic interaction between E2 and the DNA damage response (DDR) protein TOPBP1 is required for E2 segregation function. In non-infected cells, following DNA damage, TOPBP1 is recruited to the mitotic host genome via interaction with MDC1 and this interaction protects DNA integrity during mitosis. Recently we demonstrated that the E2-TOPBP1 interaction activates the DNA damage response (DDR) during mitosis independently from external stimuli, promoting TOPBP1 interaction with mitotic chromatin and therefore segregation of the viral genome. Therefore, the virus has hijacked an existing host mechanism in order to segregate the viral genome. This intricate E2 function will be described and discussed.
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pmc1 Introduction

Human papillomaviruses (HPV) are causative agents in around 5% of all cancers, including cervical and oropharyngeal cancers [[1], [2], [3], [4]]. HPV only productively infect epithelial cells and the viral life cycle is intricately related to differentiation of infected skin, with mature viruses egressing from the upper layers of infected tissue [5,6]. Infection occurs via skin abrasions, and during division of the infected cell the viral genome locates to the nucleus where viral gene expression then occurs [[7], [8], [9], [10], [11]]. The viral oncogenes E6 and E7 target tumor suppressors including p53 and pRb, respectively, resulting in enhanced proliferation of the infected cell and initiation of the differentiation process [12,13]. The viral genome is an 8 kb double stranded DNA genome that must reside in the host nucleus in order to be replicated. During infection, there is an initial phase where the genome establishes at around 50 copies per cell that is maintained through differentiation until the upper layers of the infected epithelium where viral genome amplification occurs [5]. During this phase of the viral life cycle, the structural proteins L1 and L2 are synthesized which incorporate the viral genomes into infectious particles that egress from the infected epithelium [14]. Two viral proteins, E1 and E2, are required for replication of the viral genome in association with host factors [15,16]. E2 forms homodimers and binds to 12bp palindromic sequences in the viral genome via a carboxyl-terminal domain. The amino-terminal domain of E2 interacts with E1 and recruits it to the viral replication origin (which is surrounded by E2 DNA binding sites) whereupon E1 forms a di-hexameric complex that replicates the viral genome in association with host factors [15,16]. This review will focus on the E2 protein and its role in viral genome segregation and activation of the DNA damage response (DDR).

2 Function of papillomavirus E2 proteins

As well as activating DNA replication via E1 recruitment, E2 has two other known functions during the viral life cycle. E2 regulates transcription from the host genome, creating an environment for facilitating the viral life cycle [17]. For example, E2 represses transcription of innate immune genes, and also TWIST1 [17,18]. Repression of TWIST1 prevents epithelial-to-mesenchymal transition (EMT) and alters the migration properties of E2 expressing cells [18,19]. The role of TWIST1 repression in the viral life cycle is currently being investigated in our lab. While E2 can repress transcription from integrated viral genomes, it is not clear whether it can regulate transcription from episomal genomes [[20], [21], [22], [23], [24], [25], [26], [27]]. The third role for E2 during the viral life cycle is to segregate the viral genome into daughter nuclei following mitosis [28]. Without this function, the viral genome could potentially be located in the cytoplasm following cell division, ending the viral life cycle (Fig. 1).Fig. 1 The E2 protein simultaneously binds to the viral genome and host chromatin during differentiation, promoting genome location in daughter nuclei. Failure to locate to mitotic chromatin would potentially result in viral genomes residing in the cytoplasm following cell division, ending the viral life cycle. Created using BioRender.com.

Fig. 1

3 E2 and viral genome segregation

20 years ago, the seminal paper from You and Howley demonstrated that BRD4 was an important partner protein for BPV1 (bovine papillomavirus 1) E2 interaction with mitotic chromatin [29,30]. Extensive subsequent studies investigated the role of BRD4 in the interaction of HPV E2 proteins with mitotic chromatin, with some reports suggesting that HPV E2 protein mitotic interaction is independent of BRD4 interaction [31,32], while others suggested that there was a role for BRD4 in HPV E2 interaction with mitotic chromatin [33]. For all E2 proteins, interaction with BRD4 is required for the transcriptional activation function of E2 [31,34,35] and has also been implicated in E2-mediated transcriptional repression [36] as well as regulation of E2 protein stability [[37], [38], [39]]; BRD4 is a known transcriptional regulator [40]. HPV16 E2 interaction with BRD4 is also implicated in regulation of E2 DNA replication function and is required for the maintenance of episomal viral genomes in human keratinocytes [41,42]. Until our recent work, it was unclear how HPV16 E2 (E2 from now on unless stated otherwise) segregation function is mediated, but we have demonstrated that an interaction between E2 and the cellular protein TOPBP1 is required for E2 interaction with mitotic chromatin and plasmid segregation function.

4 TOPBP1 functions

We discovered the interaction between E2 and TOPBP1 using a yeast-two hybrid screen and implicated TOPBP1 as an important viral replication factor [43,44]. TOPBP1 has 9 BRCT (BRCA1 carboxyl-terminal) domains that were originally identified as hydrophobic protein interacting pockets [45,46]. No protein has so many BRCT domains, and these provide TOPBP1 the ability to interact with a number of host proteins as a scaffold to regulate several aspects of nucleic acid metabolism in cells. A major function for TOPBP1 is activation of the ATR kinase following DNA damage, inducing a DNA damage response (DDR) signaling cascade that results in cell cycle arrest and repair of damaged DNA prior to cell cycle restart [[47], [48], [49], [50], [51]]. The activation of ATR is carried out by a conserved TOPBP1 domain that is independent from the BRCT domains [51]. TOPBP1 is also required for the initiation of DNA replication in mammalian cells via interaction with the host protein Treslin and others [[52], [53], [54], [55], [56], [57]]. We have also implicated TOPBP1 in the regulation of HPV16 replication as failure to interact with E2 disrupts E1-E2 replication, and TOPBP1 can also bind to E1 [41,58,59]. As well as regulating the DNA damage response and replication, TOPBP1 can also regulate host gene transcription via interaction with host factors including p53, E2F1 and chromatin remodeling factors [[60], [61], [62], [63], [64]]. The oligomerization state of TOPBP1 is regulated by AKT phosphorylation (a kinase activated by HPV infection), and this can alter the function of TOPBP1, switching between its known properties [[65], [66], [67], [68], [69], [70]].

More recently, TOPBP1 has been shown to be involved in several functions during mitosis [71]. It was initially shown that TOPBP1 prevents transmission of DNA damage to daughter cells by forming early mitotic foci that associate with SLX4, a structure selective nuclease, followed by damage resolution in the subsequent G1 phase [72]. TOPBP1 also recruits TOP2A to ultra-fine anaphase bridges to facilitate their resolution by promoting decatenation of entangled chromosomes [73]. It also became apparent that TOPBP1 mediates DNA processing during mitosis and promotes unscheduled DNA synthesis in order to maintain genome integrity [74,75]. Recent mechanistic studies further enhanced our understanding of TOPBP1 function during mitosis. In mitosis, DNA double strand break repair pathways are inactivated to maintain genome integrity, although early steps in the DDR process such as recruitment of MDC1 to sites of mitotic DNA damage via γH2AX interaction still occur [76,77]. CK2 phosphorylation of MDC1 promotes mitotic recruitment of TOPBP1 to MDC1 and therefore sites of DNA damage [78]. Disruption of the MDC1-TOPBP1 interaction results in mitotic radiosensitivity, development of micronuclei and chromosomal instability [78]. The TOPBP1 and MDC1 complex form a filamentous bridge between DNA DSBs, delaying repair by non-homologous end joining (NHEJ) until the following G1 phase [78]. Therefore, during mitosis TOPBP1 is involved in maintaining genome integrity and is extremely active, making it a potential candidate for mediating E2 interaction with mitotic chromatin and E2 segregation function. Fig. 2 summarizes some of the properties of TOPBP1 due to mitotic DNA damage.Fig. 2 During mitosis, DNA damage promotes MDC1 recruitment to γH2AX and MDC1 is phosphorylated by CK2 promoting the recruitment of TOPBP1. Closer association of TOPBP1 with mitotic chromatin promotes viral genome stability. Created using BioRender.com.

Fig. 2

5 TOPBP1 interaction is essential for E2 recruitment to mitotic chromatin and plasmid segregation function

Given the critical roles for TOPBP1 during mitosis, and our previous demonstration of an E2-TOPBP1 interaction, we investigated whether TOPBP1 interaction was important for E2 recruitment to mitotic chromatin and plasmid segregation function. As TOPBP1 BRCT domains interact with phosphorylated proteins [79], we mutated conserved serine and threonine residues on E2 and identified serine 23 as critical for interaction with TOPBP1 in vivo and in vitro [80]. CK2 is the kinase responsible for E2 phosphorylation on serine 23 and therefore promotes the E2-TOPBP1 complex formation [80]. Wild type E2 (E2-WT) and TOPBP1 protein levels are increased in mitotic cells (TOPBP1 levels are only increased in an E2-WT dependent manner), while an E2 mutant that failed to bind TOPBP1 (serine 23 mutated to an alanine, E2-S23A) did not stabilize either protein. E2-S23A also had aberrant interaction with mitotic chromatin when compared with E2-WT, demonstrating that E2 interaction with TOPBP1 is required for E2 recruitment to mitotic chromatin. Mutant viral genomes with an E2-S23A mutation resulted in a loss of E2 protein expression and viral genome integration and we demonstrated that stromal interaction (the presence of 3T3-J2 mouse fibroblasts that are used in organotypic rafting) results in E2 proteasomal degradation when interaction with TOPBP1 is lost [80,81]. Having established that E2 interaction with TOPBP1 is required for E2 interaction with mitotic chromatin, and the HPV16 life cycle, we next investigated whether the E2-TOPBP1 interaction was required for E2 plasmid segregation function. To do this we developed two novel assays and demonstrated that E2 interaction with TOPBP1 was required for E2 plasmid segregation function, and that CK2 disruption blocks segregation function [82]. The more robust of these assays involves labeling plasmids containing E2 binding sites with fluorescent dyes and transfecting them into stable cell lines expressing E2 or E2-S23A (compromised in TOPBP1 interaction). We can then visualize recruitment of the fluorescent plasmid to mitotic chromatin by E2 and also measure the persistence of the fluorescent plasmid over time. In addition to TOPBP1 interaction, we also demonstrated that E2 interaction with BRD4 is required for mitotic chromatin interaction and plasmid segregation function [83]. TOPBP1 and BRD4 exist in the same cellular complex and an E2 mutant defective in binding the carboxyl-terminal domain (CTM) of BRD4 (E2 R37A, arginine mutated to alanine) is still able to complex with BRD4 via interaction with TOPBP1 [83]. Fig. 3 summarizes the E2-TOPBP1-BRD4 complex interaction during mitosis.Fig. 3 E2 simultaneously interacts with both TOPBP1 and BRD4 on mitotic chromatin to promote segregation of the viral genome. Interaction with both host proteins is required for efficient E2 interaction with mitotic chromatin and for E2 plasmid segregation function. Created using BioRender.com.

Fig. 3

The results from these experiments demonstrated that E2 interaction with TOPBP1 is required for interaction with mitotic chromatin and plasmid segregation function, and that E2-WT is stabilized in mitosis in a TOPBP1 interacting dependent manner. We next investigated how E2 was stabilized at mitosis.

6 E2-TOPBP1 induces a mitotic SIRT1-p300 acetylation switch that controls protein stability

Previously, we demonstrated that SIRT1, a class III deacetylase, can deacetylate E2 and reduce its stability [84]. SIRT1 can also control the acetylation status and function of TOPBP1 [85,86]. Recently, we demonstrated that there is enhanced E2 and TOPBP1 acetylation during mitosis, and that this acetylation increases the stability of the proteins [87]. There are two conserved lysines on E2 proteins (K111 and K112 in HPV16 E2) and K111 is p300 acetylated [88,89]; we demonstrated that E2 K111 is hyperacetylated by p300 during mitosis as SIRT1 function is turned off in an E2-TOPBP1 interaction dependent manner [87]. Acetylation of E2 on K111 recruits TOP1 to E2 and protects E2 K112 from being ubiquitinated and therefore targeted for proteasomal degradation [87]. Knockdown of p300 resulted in a loss of increased E2 (and TOPBP1) mitotic acetylation and therefore increased protein levels. As well as regulating E2 and TOPBP1 stability, we also confirmed that during mitosis, the turn off of SIRT1 function results in enhanced acetylation and stability of p53, a known SIRT1 substrate [87]. We are currently investigating the role of mitotic p53 acetylation induced by the E2-TOPBP1 complex, but we have demonstrated that p53 expression is important during the HPV16 life cycle and this acetylation may be critical for this p53 function [90].

As well as p53, SIRT1 deacetylates a number of other DDR proteins involved in the viral life cycle including WRN, NBS and CHK2 [[91], [92], [93]], and SIRT1 is required for HPV life cycles [94]. We will now discuss the role of the E2-TOPBP1 complex in mitotic activation of the DDR, something that we have demonstrated [87].

7 E2-TOPBP1 interaction and mitotic activation of the DNA damage response (DDR)

The seminal paper from Moody and Laimins demonstrated that activation of ATM during differentiation is required for HPV life cycles [95]. The precise mechanism of how the DDR is activated in HPV16 positive cells has remained unclear. It has been proposed that E7 is responsible for activating the DDR, a logical extension from work demonstrating that E7 can induce genomic instability [[95], [96], [97], [98], [99], [100]]. More recently, we have discovered that viral genomes lacking E6 or E7 expression retain the ability to activate the DDR [101], and that during keratinocyte differentiation the presence of E7 is unable to activate the DDR, while cells retaining the intact HPV16 genome (and E2 expression) are able to do this [80]. Overexpression of E1 is able to induce DNA damage indicating that E1 is also a candidate for DDR activation [[102], [103], [104], [105]]. We have also proposed that torsional stress on the replicating viral genome could induce the DNA DDR in HPV16 infected cells [106,107]. However, in our recent report, we demonstrate that the E2-TOPBP1 interaction strongly activates the DDR during mitosis [87]. Increased CHK2 acetylation due to E2-TOPBP1 turnoff of SIRT1 function results in enhanced CHK2 phosphorylation, which activates CHK2 enzyme activity [87,93]. Not only that, using a pS/TQ specific antibody (that recognizes ATM/ATR substrates) we demonstrated that there is global DDR activation during mitosis only in E2-WT cells; failure to bind TOPBP1 abrogates DDR activation [87]. E2-TOPBP1 activation of the DDR would promote a closer association of the viral genome with the mitotic chromatin, as DDR activation results in TOPBP1 recruitment to mitotic chromatin via MDC1 interaction [78]. Therefore, the virus is boosting its segregation function by mitotic activation of the DDR, as it is promoting closer association of the viral genome with mitotic chromatin. We could not detect any activation of the DDR in human foreskin keratinocytes immortalized only by E6/E7 [87]. We propose that the major activator of the DDR during the viral life cycle is due to E2-TOPBP1 interaction. Strong DDR activation is only detected in HPV16 positive keratinocytes following differentiation (not in E6/E7 expressing cells) and we are currently investigating whether the mitotic E2-TOPBP1 induced DDR can be permanently switched-on following differentiation. It has been proposed that HPV16 infected cells generate a pseudo G2/M like cell cycle phase during differentiation, which would support the idea that the mitotic E2-TOPBP1 DDR activation could be active during differentiation [12]. It is striking that when organotypic raft cultures are stained with γH2AX, almost every cell is positive in HFK containing episomal HPV16 genomes, but not in HFK with integrated genomes that have lost E2 expression but retain robust E7 expression [80]. This supports the hypothesis that E2 is required for DDR activation during the viral life cycle. Fig. 4 summarizes our confirmed data about E2 mitotic activation of the DDR.Fig. 4 Mitotic E2-TOPBP1 interaction activates the DDR. During mitosis, the E2-TOPBP1 interaction switches off SIRT1 function, enhancing the acetylation and stability of viral and host proteins. This contributes to activation of CHK2 phosphorylation by ATM ultimately promoting interaction of the E2-TOPBP1-viral genome complex with the mitotic chromatin. Created using BioRender.com.

Fig. 4

8 Conclusions and future directions

We have mechanistically determined two main properties for the E2-TOPBP1 interaction during the HPV16 life cycle: it is required for viral genome segregation and also for activation of the DDR. It is of course possible that the viral oncogenes could amplify the E2-TOPBP1 induced DDR, but we propose that the initiation of DDR activation by HPV16 requires the E2-TOPBP1 interaction. We are currently investigating whether E2-TOPBP1 mitotic DDR activation persists in differentiating cells. The E2-TOPBP1 complex also potentially generates a genomic vulnerability in HPV16 infected cells. We have demonstrated that the expression of E2 promotes genomic instability and sensitivity to the DNA damaging agent cisplatin [108]. Our preliminary studies have demonstrated that the E2-TOPBP1 interaction is required for the cisplatin induced sensitivity by E2. It is striking that E2 expression in HPV16 positive head and neck cancers results in better overall survival, presumably because these tumors respond better to therapeutic regimes that include cisplatin and radiation [109,110]. Much work remains to be done. We must determine how E2-TOPBP1 interaction during mitosis (and likely during the viral life cycle in differentiating keratinocytes) is activating the DDR, and how the E2-TOPBP1 interaction is disrupting SIRT1 function and binding partners during mitosis. We are currently investigating this. The E2-TOPBP1 mitotic complex also provides potential targets for disrupting viral genome segregation, which would be antiviral and also potentially be therapeutically advantageous in HPV positive cancers that retain E2 expression. We have already demonstrated that the CK2 inhibitor CX4945 can disrupt the E2-TOPBP1 complex, and that CK2 is required for E2 plasmid segregation function [82]. SIRT1 agonists and antagonists may also disrupt the function of the E2-TOPBP1 complex, while disruption of BRD4 function may also abrogate E2 interaction with mitotic chromatin. All of these approaches are currently being investigated in our lab.

CRediT authorship contribution statement

Apurva T. Prabhakar: Writing – review & editing, Writing – original draft, Conceptualization. Iain M. Morgan: Writing – review & editing, Writing – original draft, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.

Acknowledgements

This work was supported by 10.13039/100018390 US 10.13039/100000002 NIH grant R01DE029471 (10.13039/100011778 IMM ) and R21AI178143 (10.13039/100011778 IMM ).
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