DNA damage response is hijacked by human papillomaviruses to complete their life cycle

Abstract

The DNA damage response (DDR) is activated when DNA is altered by intrinsic or extrinsic agents. This pathway is a complex signaling network and plays important roles in genome stability, tumor transformation, and cell cycle regulation. Human papillomaviruses (HPVs) are the main etiological agents of cervical cancer. Cervical cancer ranks as the fourth most common cancer among women and the second most frequent cause of cancer-related death worldwide. Over 200 types of HPVs have been identified and about one third of these infect the genital tract. The HPV life cycle is associated with epithelial differentiation. Recent studies have shown that HPVs deregulate the DDR to achieve a productive life cycle. In this review, I summarize current findings about how HPVs mediate the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and RAD3-related kinase (ATR) DDRs, and focus on the roles that ATM and ATR signalings play in HPV viral replication. In addition, I demonstrate that the signal transducer and activator of transcription-5 (STAT)-5, an important immune regulator, can promote ATM and ATR activations through different mechanisms. These findings may provide novel opportunities for development of new therapeutic targets for HPV-related cancers.

1. Introduction

DNA damage occurs naturally in cells and is associated with intrinsic reagents such as reactive oxygen species (ROS) or extrinsic agents such as ultraviolet (UV), ionizing radiation (IR), and chemotherapeutic drugs. The process begins with an alteration in the structure of DNA through formation of single and double DNA breaks, as well as other changes. In response to DNA damage, cells signal through a complex network. This response plays important roles in processes such as genome stability, tumor transformation, and cell cycle regulation (Matt and Hofmann, 2016). To maintain genome stability, cells must repair damage-induced DNA lesions. If the damage cannot be fixed, cells activate cell cycle check points to trigger cell death. Previous studies have shown that deregulation of the DNA damage response (DDR) can contribute to the development of various cancers such as hereditary non-polyposis colorectal cancer, non-small-cell lung cancer, breast cancer, and ovarian cancer, as well as other cancer-causing conditions such as Fanconi anaemia (FA), xeroderma pigmentosum, and ataxia telangiectasia (Lord and Ashworth, 2012; Sperka et al., 2012; O'Connor, 2015; Seebode et al., 2016). Many small molecule modulators targeting the DDR have been proposed as potential therapeutic treatments against cancers ranging from acute myeloid leukaemia to lung squamous cell carcinoma, as well as cervical squamous cell carcinoma (Pearl et al., 2015).

Worldwide, cervical cancer is the fourth most common cancer in women and the second most frequent cause of cancer-related death. Human papillomaviruses (HPVs) are the causative agents of cervical cancer as well as other anogenital cancers (Zur Hausen, 2002). There are more than 200 types of HPVs. Around 40 of them infect the genital tracts and are sexually transmitted. HPVs can be classified as high-risk or low-risk according to their association with cancers. Infection by low-risk HPVs can lead to cutaneous lesions or benign mucosal lesions while high-risk HPV infection may develop into anogenital or oropharyngeal cancers (Zur Hausen, 2009; Doorbar et al., 2012). Evidence suggests that persistent infection of high-risk HPVs causes genetic instability (Ho et al., 1995; Herrero et al., 2000) and DNA damage repair machinery is utilized by HPVs for productive viral replication (Hong and Laimins, 2013b; Galloway and Laimins, 2015; McKinney et al., 2015). The purpose of this review is to understand better how HPVs employ the DDR, especially ataxia-telangiectasia mutated kinase (ATM)/ATM-and RAD3-related kinase (ATR) signaling, in their life cycle. Before describing what is known about the pathway, I will summarize the cellular pathways involved in the DDR and give a general description of mechanisms that have been reported to be involved in regulating the productive life cycle of HPVs. Then I will discuss the relationship between the DDR and the HPV viral life cycle in detail, including the roles of the signal transducer and activator of transcription-5 (STAT-5).

2. DNA damage response

2.1. Significance and classification of DNA damage response

It has been estimated that up to 200 000 DNA damage lesions occur naturally in each cell every day (Atamna et al., 2000). The causes of DNA damage can be either endogenous or environmental. Endogenous causes of DNA damage include replication errors, unrepaired single strand lesions, and base deamination or loss (Curtin, 2012). Environmental causes result mainly from exposure to damaging agents, including UV, IR, ROS, S-adenosyl methionine, dietary nitrosamines, and tobacco. In response to DNA damage, cells mount collective cellular events such as cell-cycle arrest, regulation of gene expression and DNA replication, activation of DNA damage repair, and cell fate decisions (Matt and Hofmann, 2016). Proper DNA damage repair is necessary for genome stability, prevention of transformation, and tumor suppression. Deregulation of the DDR leads to many clinically relevant diseases such as premature aging, neurodegenerative disorders, and cancer formation (Jackson and Bartek, 2009).

DNA damage often results in DNA mutations, crosslinking, and single-strand breaks (SSBs) as well as double-strand breaks (DSBs). Damaged cells activate various pathways to repair these lesions, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair, non-homologous end joining (NHEJ), homologous recombination repair (HRR), and interstrand crosslink repair (Curtin, 2012). Among these, NHEJ and HRR are the two major repair mechanisms for DSBs in eukaryotes, BER enzymes are major players for SSB repair (Dianov and Hubscher, 2013), and NER works for other bulk single-strand lesions. During these processes, various complexes of cellular proteins are recruited to DNA damage loci to activate DNA damage-responsive phosphatidylinositide 3 kinase (PI3K)-like Ser/Thr kinases, which consist of ATM, ATR, and DNA-dependent protein kinase catalytic subunit kinase (DNA-PKcs). For example, the heterotrimeric meiotic recombination 11 (Mre11)/Rad50/Nijmegen breakage syndrome protein 1 (NBS1) (MRN) complex (Carney et al., 1998) and the Ku70/Ku80 complex (Cary et al., 1997) are responsible for repairing DSBs. When cells experience SSBs, the Rad9-Hus1-Rad1 (9-1-1) complex is activated to facilitate recruitment of the replication protein A (RPA) and other factors. The functions of ATM and ATR (Fig. 1) will be addressed in detail below.

Fig. 1.

Signaling pathways of ATM and ATR

The DNA damage response is activated by ATM, ATR, and DNA-PKcs. All three play central roles in DDR. The ATM-CHK2 and ATR-CHK1 signaling pathways activate the HRR. The ATM and ATR pathways can be activated respectively by DSBs and SSBs. ATM activation can be regulated by Tip60, MRN complex, ATR, and PP2A as well as Wip1. The activated ATM can further trigger the activation of CHK2, SMC-1, FANCD2, BRCA1, as well as H2AX. In addition, the activated CHK2 can phosphorylate Cdc25A and p53. The ATR pathway can be activated by ATRIP, TopBP1, claspin, and 9-1-1 complex. The activation of ATR leads to phosphorylation of various downstream targets such as CHK1, SMC-1, ATM, and p21. Furthermore, CHK1 can facilitate phosphorylation of Wee1, RAD51, Cdc25A, p65, and Rb

2.2. Function and regulation of ATM

ATM function and activation in the DDR depend on its phosphorylation. When cells encounter DNA damage signals, the MRN complex provides the platform for inactivated ATM to be phosphorylated (Lee and Paull, 2004). Several other cellular factors are also necessary to activate ATM directly or indirectly, such as Tat interactive protein 60 (Tip60) (Sun et al., 2005), poly adenosine diphosphate (ADP)-ribose polymerase (PARP) (Aguilar-Quesada et al., 2007), and phosphatase and tensin homolog (PTEN) (Zhang R. et al., 2016). A number of factors can attenuate ATM phosphorylation including the serine/threonine protein phosphatases such as PP2A, Wip1, and PP5 (Ali et al., 2004; Goodarzi et al., 2004; Shreeram et al., 2006). The activated ATM kinase phosphorylates Kruppel-associated box (KRAB)-associated protein-1 (White et al., 2006), releases this substrate from the DNA lesion, and facilitates the recruitment of MRE11 and C-terminal binding protein (CtBP)-interacting protein (Ziv et al., 2006; Goodarzi et al., 2008). In addition, ATM activation leads to the formation of RAD51 nucleofilaments and the formation of RAD51/γ-H2AX foci (Bakr et al., 2015). The histone variant H2AX, which spreads to sites and DNA breaks, can be also phosphorylated by ATM (Burma et al., 2001).

ATM activation is important for the regulation of cell cycle checkpoints and downstream pathways. For the G1/S checkpoint, ATM triggers a p53-independent checkpoint kinase 2 (CHK2)/Cdc25A arm (Falck et al., 2001) and an MDM2/p53 arm (Maya et al., 2001). S-phase checkpoint control depends on the activation of NBS1, breast cancer type 1 susceptibility protein (BRCA1) and Fanconi anemia group D2 protein (FANCD2) (Gatei et al., 2001; D'Amours and Jackson, 2002; Taniguchi et al., 2002). ATM activation inhibits Cdc25 phosphatase resulting in S/G2 arrest (Matsuoka et al., 1998). G2/M arrest can be mediated by BRCA1 or p53 in a Cdc2-or Cdc25C-dependent manner. The downstream effector kinase CHK2 activation triggers Cdc25C phosphorylation causing a G2/M arrest (Zhou et al., 2000). In addition, ATM mediates p53 phosphorylation and stabilization to regulate cyclin-dependent kinase 2 (CDK2)/cyclin E-dependent G1 cell cycle arrest (Canman et al., 1994; Banin et al., 1998). Many substrates, including γ-H2AX (Fernandez-Capetillo et al., 2004), the cohesin factor structural maintenance chromosome-1 (SMC-1) (Kitagawa et al., 2004), and CHK2 (Bouwman and Jonkers, 2012), are recruited to the sites of damage. As a consequence, CHK2 is activated, which then phosphorylates additional substrates such as BRCA1 tumor suppressor (Cortez et al., 1999), p53 tumor suppressor (Chen et al., 2005), and the Cdc25 family of phosphatases (Blasina et al., 1999).

2.3. Function and regulation of ATR

In parallel to ATM signaling, ATR signaling is critical to HRR in response to SSBs. ATR is known to be important to cell survival and its inactivation in mice by disruption of the kinase domain results in early embryonic lethality (de Klein et al., 2000). Furthermore, a splicing mutation of ATR leads to Seckel syndrome (O'Driscoll et al., 2003). In response to DNA damage stimuli, long stretches of single strand DNA (ssDNA) are generated, which are coated with RPA (Coverley et al., 1992). ATR is recruited to RPA-ssDNA complex and interacts with its canonical partners: ATR-interacting protein (ATRIP) (Zou and Elledge, 2003), claspin (Kumagai and Dunphy, 2003) and topoisomerase IIβ-binding protein 1 (TopBP1) (Kumagai et al., 2006). Among these, TopBP1 interacts with the 9-1-1 complex, which is recruited to ssDNA lesions. ATR kinase activities can be regulated by several factors such as ATRIP (Zou and Elledge, 2003) and TopBP1 (Kumagai et al., 2006). Once activated, ATR phosphorylates various downstream substrates including BRCA1 (Chen, 2000), MCM proteins (Cortez et al., 2004), and checkpoint kinase 1 (CHK1) (Liu et al., 2000). In turn, CHK1 phosphorylates Cdc25A (Falck et al., 2002), Wee1 kinase (Lee et al., 2001), and RAD51 (Sorensen et al., 2005).

The ATR pathway can be activated not only by SSBs but also by DSBs. Recent studies have provided evidence that the ATR and ATM pathways are interlinked (Smith J. et al., 2010). For example, ATM phosphorylation is necessary for a rapid activation of ATR when cells are exposed to DSBs (Byun et al., 2005; Myers and Cortez, 2006). ATR activation by radiation-stimulated DSBs shares a common pattern of induction of an S/G2 phase arrest with ATM signaling (Jazayeri et al., 2006; Walker et al., 2009). In addition, activation of ATR signaling depends on the MRN complex that is also necessary for ATM signaling (Yarden et al., 2002; Myers and Cortez, 2006). Both the ATM and ATR pathways can be manipulated by various viruses, such as Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and HPV (McFadden and Luftig, 2013; Hollingworth and Grand, 2015). The regulation of the ATM/ATR pathways by HPV will be discussed later.

3. Life cycle of HPVs

3.1. HPV life cycle

HPVs are the major etiological agents responsible for cervical cancer (Zur Hausen, 2002). In recent years HPVs have been shown also to be involved in many other genital cancers such as those of the vulva, vagina, anus, penis, and oral cavity. About nine types of HPVs are considered as high-risk types, including HPV16, HPV18, HPV31, and HPV45, that are the causative agents of most anogenital cancers. The low-risk types, including HPV6 and HPV11, can cause genital warts. The life cycle of HPVs is dependent on epithelial differentiation (Fig. 2). During infection, HPVs escape immune surveillance and can remain latent for decades. HPVs infect stratified squamous epithelia by entering the cells in the basal layer that becomes exposed following trauma or wounding (Hebner and Laimins, 2006; Doorbar et al., 2012). The infected basal cell divides into a new basal cell and a daughter cell that migrates to the upper layers of the epithelium as it undergoes terminal differentiation. HPV episomes are replicated and equally distributed to the new basal cell and the daughter cell.

Fig. 2.

Life cycle of human papillomaviruses

HPVs infect basal layer keratinocytes when the basal layer of stratified epithelia is exposed to the virus. On the left, a normal uninfected epithelium is shown for the regular differentiation. On the right, the HPV-infected epithelium is shown with the progress of HPV viral proteins expression. After persisting infection, HPVs replicate with cellular chromosomes in basal cells. Upon the differentiation, more viral genes are observed in differentiated cells. The late gene expression and viral replication are activated, followed by virion assembly and release of newly synthesized virions from the top layers of epithelium

HPVs maintain low copy numbers of viral genomes (around 100 copies per cell) in undifferentiated cells. Upon differentiation, the viral copy number increases rapidly to levels of around 1000 copies per cell in a process referred to as amplification. This is followed by virion assembly and viral release from the upper layers of the epithelium (Longworth and Laimins, 2004).

3.2. HPV viral protein function

The genomes of HPVs are about 8 kb in size and encode multiple viral gene products, including E1, E2, E4, E5, E6, E7, as well as L1 and L2 (Hebner and Laimins, 2006). E1 and E2 are two genes that regulate initiation of HPV genome replication (Sedman and Stenlund, 1995; McBride, 2013), whereas E4 and E5 regulate late viral functions (Dimaio and Petti, 2013). E6 and E7 are necessary for HPV genome maintenance and amplification, and act as two oncogenes to alter the host environment to be advantageous for viral replication (Thomas et al., 1999; Munger and Howley, 2002; Wise-Draper and Wells, 2008; Wallace and Galloway, 2015). L1 and L2 are the two capsid proteins synthesized following productive replication (Hebner and Laimins, 2006; Thomas et al., 2008).

HPV viral proteins function cooperatively to regulate the HPV life cycle. The E1 protein acts as a DNA helicase/ATPase to facilitate DNA unwinding and recruits host DNA polymerases to viral origins (Hughes and Romanos, 1993; Conger et al., 1999). E2 has DNA-binding activities and is important for DNA segregation in mitotic cells (Oliveira et al., 2006; Poddar et al., 2009). Bromodomain-containing protein 4 (Brd4) has been implicated in this E2 function, and the interaction between Brd4 and E2 is also required for E2-dependent transcriptional regulation (McPhillips et al., 2006; Wu et al., 2006). In addition, E2 regulates expressions of E6, E7, E1, as well as E2 itself via controlling transcription of the early viral promoter (Steger and Corbach, 1997). E8^E2C is a truncated form of E2 that inhibits early gene expression and viral replication (Stubenrauch et al., 2000). E1 and E2 work cooperatively in the initiation of viral replication (Frattini and Laimins, 1994; Sedman and Stenlund, 1995). E6 and E7 promote genome maintenance and viral replication (Cheng et al., 1995; Thomas et al., 1999) in addition to their roles in cell transformation and immortalization. An HPV31 genome containing an E6 or E7 mutation is not able to be maintained stably as an episome (Thomas et al., 1999). The high-risk E6 binds to the cellular E3 ubiquitin ligase E6-associated protein (E6AP) to degrade p53 (Scheffner et al., 1990; 1993; Huibregtse et al., 1991) and to inhibit p53 function by blocking acetylation (Hebner et al., 2007) in a p300-dependent manner (Patel et al., 1999). The E7 protein binds to pRb to facilitate the regulation of cell cycle events by E2F family members (Dyson et al., 1989; Munger et al., 1989; Longworth et al., 2005). E5 has been shown to be involved in cell motility, adhesion, and proliferation (Kivi et al., 2008; Liao et al., 2013). L1 and L2 capsid proteins are responsible for viral chromatin packaging and virion assembly (Nelson et al., 2002; Darshan et al., 2004). The lack of viral DNA polymerases and other necessary factors leads HPVs to rely on host factors to accomplish genome amplification.

4. Regulation of HPV viral replication

4.1. Classification of regulators for HPV viral replication

HPV viral replication is dependent on epithelial differentiation, and is regulated by viral factors, such as E1 and E2 (Kadaja et al., 2009), and host factors. Because HPVs do not encode their own DNA polymerases and other factors, their replication is largely dependent on host factors such as transcriptional factors, microRNAs (miRs), kinases, apoptotic caspases, epigenetic enzymes, and DNA damage signaling (Moody and Laimins, 2010; Kajitani et al., 2012; Hong and Laimins, 2013b). Before describing the interaction between the HPV life cycle and the DDR, I will provide a general description of what is known about the cellular factors involved in HPV viral replication, though the actual process of amplification is still not clear. The regulation of HPV viral replication by the DDR will be then discussed in detail in the following section.

4.2. Cellular enzymes

Several of the host factors that are important for HPV viral replication have been identified as cellular enzymes associated with the DDR. For example, the ubiquitin-specific protease 1 (USP1)-associated factor 1 (UAF1)-USP deubiquitinase complex, which is suggested to be associated with the DDR (Chen et al., 2011), can be recruited by E1 to facilitate HPV DNA replication (Lehoux et al., 2014). The activated CDK2 phosphorylates NBS1 (Wohlbold et al., 2012) and mediates HPV genome maintenance (Fradet-Turcotte et al., 2010). The activity of caspase-3 is required for HPV genome replication (Moody et al., 2007), and caspase-3-dependent apoptosis can be inhibited by ATR/CHK1 (Myers et al., 2009). Activation of the nuclear factor-(B (NF-(B) pathway has been implicated in HPV genome amplification (Nakahara et al., 2015; Satsuka et al., 2015) and can be induced by DNA damage (Janssens and Tschopp, 2006). The deregulation of these factors suggests that HPV induces the DDR while suppressing cellular apoptotic events. Furthermore, binding of histone deacetylases (HDAC) to E7 contributes to E7-facilitated HPV genome amplification (Longworth et al., 2005) and HDACs have been reported to function in the DDR (Miller et al., 2010; Thurn et al., 2013). The deacetylase sirtuin 1 facilitates HPV DNA replication, partly by modulating histone acetylation (Langsfeld et al., 2015). In addition, repression of Brd4, which insulates chromatin from DNA damage signaling (Floyd et al., 2013), deregulates E2-dependent HPV oncogene expression (Smith J.A. et al., 2010). The association of these factors with HPV genomes further indicates that the DDR is involved in HPV viral replication.

4.3. Transcription factors

Cellular transcription factors also play a role in HPV viral replication. Some, including YY-1 (Ai et al., 2000), TATA-binding protein (TBP) (Hartley and Alexander, 2002), activator protein 1 (AP-1) (Offord and Beard, 1990), Oct-1 (O'Connor and Bernard, 1995), and Sp1 (Stunkel and Bernard, 1999), act on the HPV early promoters located upstream of the E6 open reading frame (ORF). Others, such as CCAAT/enhancer binding protein β (C/EBPβ) isoforms, liver-enriched inhibitory protein (LIP), and liver-enriched transcriptional activator protein (LAP) (Gunasekharan et al., 2012), act on the late promoter. Several of these transcription factors have been shown to be related to the DDR. For example, YY-1 is essential in homologous recombination-based DNA repair (Wu et al., 2007). Downregulation of the subunit of TBP is responsible for DNA damage-induced repression of RNA polymerase III transcription (Ghavidel and Schultz, 2001). Activation of Oct-1 can be induced by DNA damage (Zhao et al., 2000). The phosphorylated Sp1 colocalizes with (-H2AX and depletion of Sp1 inhibits the repair of DSBs (Beishline et al., 2012). In addition, members of the STAT family can be differently regulated by HPVs to facilitate viral replication. Inhibition of STAT-1 by E6 and E7 is necessary for HPV genome amplification (Hong et al., 2011). In contrast, STAT-5 is activated by E7 and this activation is important for HPV genome amplification (Hong and Laimins, 2013a). Another transcription factor, Kruppel-like factor 13, can regulate STAT-5 expression, and is important for the differentiation-dependent HPV life cycle (Zhang W. et al., 2016). The relationship between STAT-5 and the DDR will be discussed later.

4.4. MicroRNAs

miRs are among other cellular factors involved in HPV viral replication. The miRs are noncoding regulatory RNAs, 18–25 nucleotides in size. They post-transcriptionally regulate mRNA stability and translation and have been reported to be associated with the DDR (Wan et al., 2011; Wang and Taniguchi, 2013). miRs are also associated with cervical cancer (Pedroza-Torres et al., 2014) and can be considered as biomarkers for high-risk HPV infection (Wang et al., 2014). HPV E7 down-regulates miR-203 and this suppression is required for HPV genome amplification through regulation of p63 (Melar-New and Laimins, 2010) and ATM DDR (Mighty and Laimins, 2011). miR-145, which is also suppressed by E7 and targets E1 ORFs, is important for HPV genome amplification (Gunasekharan and Laimins, 2013). Ectopic expression of miR-125b suppresses HPV gene expression (Nuovo et al., 2010) possibly due to the sequence homology between HPV16 L2 and miR-125b. The role of these HPV-related miRs in the DDR is still not clear.

5. HPV and DDR

5.1. HPV regulates the DDR

Upon DNA damage, many host repair factors are recruited to the damage loci to repair single or double strand breaks. HPVs hijack this repair machinery, by both inhibiting and activating the DDR, to replicate viral genes (Hong and Laimins, 2013b; Wallace and Galloway, 2014; 2015; McKinney et al., 2015). For example, HPV oncogenes have been suggested to abrogate radiation-induced DDRs (Song et al., 1998), and recent studies support the idea that HPVs regulate the DDR to mediate their life cycle. HPV E7 activates the FA repair pathway, enhances FANCD2 foci, and recruits FANCD2 and BRCA2 to chromatin (Spardy et al., 2007). Both E6 and E7 interact with BRCA1 to inhibit its transcriptional regulation (Zhang et al., 2005). HPV E6 disrupts p53 signaling (Thomas and Chiang, 2005) and interferes SSBs through interactions with XRCC1 and O⁶-methylguanine-DNA methyltransferase (Iftner et al., 2002; Srivenugopal and Ali-Osman, 2002).

Besides regulation of the DDR proteins mentioned above, HPVs are capable of manipulating host genomic destabilization. E6 and E7 can induce DNA damage independently by causing host chromosome instability (Duensing and Munger, 2002), and collaboratively by uncoupling centrosome duplication from the cell division cycle (Duensing et al., 2000). Delocalization of the microtubule motor dynein by E7 results in failed chromosome alignment (Nguyen et al., 2008). The expression of E7 results in polyploidy by inducing HPV rereplication in response to DNA damage (Fan et al., 2013). In addition, E1 and E2 act as recruiters to facilitate colocalization of the DDR proteins with the HPV replication complex, which will be addressed below.

5.2. HPV regulates ATM and ATR pathways

The two major arms of the DDR are the ATM/CHK2 and ATR/CHK1 pathways. Little is known about how HPVs employ these two arms for the viral life cycle. ATM activation can be induced by high-risk HPV E1 and E7 proteins. For example, HPV18 E1 could cause DSBs and lead to the induction of an ATM-dependent signaling cascade (Reinson et al., 2013). The HPV31 E7 oncoprotein binds to ATM and induces phosphorylation of ATM and CHK2 (Moody and Laimins, 2009). Similarly, HPV18 E7 induces elevated expression of phosphorylated ATM, as well as CHK2 and c-Jun N-terminal kinases (JNKs) (Banerjee et al., 2011). The activated ATM and CHK2 are recruited to the DNA repair centers that are colocalized with the HPV integrated replication sites (Kadaja et al., 2009), suggesting a critical role of ATM signaling in recruitment of HPV DNA to the DNA repair centers to start viral DNA synthesis.

Less is known about the effect of the viral proteins on ATR activity. Several contradictory results have been reported by different groups. An elevation of ATR protein levels was indicated in HPV16 E7-expressing cells (Spardy et al., 2008), whereas other studies showed that HPV16 E7 attenuates CHK1 phosphorylation by increasing claspin turnover (Spardy et al., 2009). Activation of the ATR pathway is induced by HPV18 E1 and E2, but not E6 or E7 (Reinson et al., 2013). However, recent studies have shown that HPV31 E7 increases ATR phosphorylation as well as CHK1 phosphorylation (Hong et al., 2015b). The levels of phosphorylated CHK1 are also enhanced and sustained in HPV16 E6-expressing fibroblasts (Chen et al., 2009). The complexity of ATR regulation by high-risk HPV proteins indicates that differences between HPV species might lead to different modulation of ATR activation and that HPV proteins employ different mechanisms to regulate ATR and ATM.

Unlike high-risk α HPV types, β HPV E6 reduces ATM protein levels (Wallace et al., 2013) and abrogates ATR activities (Wallace et al., 2012). The reduction of ATR protein levels can be explained by the degradation of p300 (Wallace et al., 2012), which is regulated by protein kinase B (PKB/AKT) (Howie et al., 2011). Furthermore, β HPV E6 inhibits the stability of p53 (Wallace et al., 2014) and attenuates BRCA1 and BRCA2 expression as well as foci formation (Wallace et al., 2015). The suppressive effect of β E6 on ATM/ATR signaling may reflect an increased cellular tolerance of DNA lesions and a reduced induction of apoptosis by the DDR. It is not known how this regulation contributes to β HPV genome amplification.

E1 and E2, instead of modulating the phosphorylation of factors of the ATM/ATR pathway, are parts of the viral replication complex that colocalize with these factors (Kadaja et al., 2009). Several cellular replication factors colocalize with HPV18 E1 in HeLa cells, including RPA, proliferating cell nuclear antigen (PCNA), and death-associated protein (DAXX). HPV18 E1 also recruits the MRN and Ku70/Ku80 complexes to the viral replication complex, and induces ATM/CHK2 activation. The same study provided some evidence that E1 is colocalized with ATRIP and CHK1. Another set of DDR proteins, including (-H2AX and 53BP1, colocalizes with the HPV DNA foci (Gillespie et al., 2012). It has been suggested that the function of E2 is to facilitate the translocation of the complex of E1 and the associated DDR proteins to the nuclear foci (Sakakibara et al., 2011). In addition, Boner et al. (2002) showed that HPV16 E2 interacts with TopBP1, and that this interaction enhances the ability of E2 to activate transcription and replication. All these findings suggest that interactions of the DDR proteins and E1/E2 play an important role in the aggregation of the HPV viral replication complex and DNA damage repair factors in the nuclear foci.

5.3. ATM and ATR DDR are necessary for HPV viral replication

The fact that many DDR proteins are deregulated by HPVs suggests that the DDR plays a significant role in HPV genome amplification. This is supported by the finding that activation of the ATM pathway is necessary for HPV genome amplification (Moody and Laimins, 2009). The same study also showed that inhibition of ATM activities has no effect on HPV genome maintenance. NBS1, as one component of the MRN complex that facilitates ATM activation, is also required for HPV genome amplification (Anacker et al., 2014). Consistently, BRCA1, as a downstream target of ATM, is important for HPV genome amplification (Chappell et al., 2016). Another downstream target, SMC-1, is activated by HPV and is required for HPV genome amplification (Mehta et al., 2015), whereas loss of FANCD2 stimulates HPV replication (Hoskins et al., 2012). In addition, RAD51 plays essential roles in the DDR and binds to HPV31 genomes. Depletion of RAD51 or inhibition of RAD51’s recombinase activity abolishes HPV viral replication upon differentiation (Chappell et al., 2016). HPV’s association with Brd4 leads to an increase in the rate of asynchronous DNA replication (Jang et al., 2014).

Compared to the ATM pathway, the role of ATR signaling for HPV replication is less well known. HPV18 transient replication induces an accumulation of ATR signaling, indicating a role of ATR in the initiation of HPV18 replication (Reinson et al., 2013). Recent studies have shown that inhibition of ATR activities reduces HPV genome amplification, and that inhibition of CHK1 activities suppresses HPV genome amplification (Hong et al., 2015b). Edwards et al. (2013) also showed that inhibition of CHK1 significantly reduces HPV episome levels in undifferentiated cells. In addition, knockdown of TopBP1, which is upstream of the ATR activation, suppresses HPV viral replication (Hong et al., 2015b). The interaction between TopBP1 and E2 also contributes to HPV genome amplification (Donaldson et al., 2012). Taken together, both ATM signaling and the ATR pathway are important for HPV viral replication.

5.4. STAT-5 activation promotes both ATM and ATR signaling for HPV viral replication

Although there is mounting evidence that the DDR is important for HPV genome amplification, less is known about how HPV regulates it to accomplish its life cycle. Recent studies investigated the relationships between HPVs and the immune response, and found that HPVs might utilize immune factors to promote the DDR by deregulation of the STAT family that contains important regulators of the innate immune response (Beglin et al., 2009; Stanley, 2012; Hong and Laimins, 2013b).

STAT signaling is part of the interferon (IFN) pathway. Canonically, IFN-α or IFN-β binds to a heterodimeric transmembrane receptor termed IFN ax-receptor (IFNAR) (Abbas et al., 2014) and activates the receptor-associated Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), resulting in the phosphorylation of inactive STAT proteins translocating from the cytoplasm to the nucleus and the induction of hundreds of genes that act to block viral propagation. The STAT family consists of STAT-1, -2, -3, -4, -5, and -6. Upon phosphorylation, STAT-1 forms a complex referred to as IFN-stimulated gene factor (ISGF3) along with STAT-2 and IFN regulatory factor (IRF)-9. The ISGF3 complex binds to the IFN stimulated elements (ISRE) located in promoter regions of many IFN-stimulated genes (ISGs) to induce their expressions. Similarly, unphosphorylated STAT-5 becomes activated following binding of cytokines to the cytokine receptors resulting in the formation of either homo-or heterodimers between its two isoforms STAT-5α and STAT-5β, and translocation from the cytoplasm to the nucleus (Ferbeyre and Moriggl, 2011). STAT-1 can induce many ISGs such as IFN-inducible double-stranded RNA-dependent protein kinase (PKR) and IFN-induced protein with tetratricopeptide repeats (IFIT) (Diamond and Farzan, 2013), while STAT-5 activates a different set of downstream genes (Doan et al., 2008). STAT-5 activities are important for the development and survival of lymphocytes (Heltemes-Harris and Farrar, 2012), as well as epithelial cells (Groner, 2002).

Evidence suggests that the STAT proteins are associated with the DDR. For example, STAT-1 is suggested to confer resistance to DNA damage (Cheon et al., 2013). STAT-3 has been shown to disrupt ATR/CHK1 signaling (Koganti et al., 2014) and phosphorylation of STAT-3 requires ATM activation (Zhang et al., 2003). STAT-5 shares 50% homology with STAT-1. Knockout mice lacking STAT-5 exhibit a perinatal lethal phenotype (Cui et al., 2004) and severely impaired lymphoid development (Yao et al., 2006). Over-expression of STAT-5 promotes the DDR and is associated with CHK2 activity (Eilon and Barash, 2011). This suggests that HPV might regulate the STAT proteins to promote the DDR.

Before discussing the relationship between the STAT proteins and the DDR, I will discuss the expression of the STAT family in HPV-positive cells. The Laimins group has shown that HPV specifically suppresses the expression of STAT-1, but not STAT-2 (Hong et al., 2011) or STAT-3 (unpublished). In contrast, HPV induces the constitutive activation of STAT-5 while only minimally affecting total levels (Hong and Laimins, 2013a). HPVs can also deregulate STAT expression by controlling the IRF transcription factors and synthesis of IFNs. For example, IRF-1 expression can be downregulated by HPV16 E7 (Park et al., 2000) and HPV38 E6E7 (Cordano et al., 2008). HPV16 E6 binds to IRF-3 to inhibit its transcriptional activity (Ronco et al., 1998). Consequently, IRF deregulation by HPV proteins leads to a reduced expression of IFN-α (Chang and Laimins, 2000), IFN-β (Ronco et al., 1998; Park et al., 2000), and IFN-κ(Rincon-Orozco et al., 2009). Among these, IFN-β has been shown to be associated with the DDR (Moiseeva et al., 2006; Cheon et al., 2013).

Hong and Laimins (2013a) have shown that inhibition of STAT-5 phosphorylation suppresses HPV genome amplification and late gene expression upon epithelial differentiation. This STAT-5-dependent regulation can be explained by two DDR mechanisms: the ATM (Hong and Laimins, 2013a) and the ATR pathways (Hong et al., 2015b) (Fig. 3). Inhibition of STAT-5 suppresses the phosphorylation of ATM, CHK2, ATR, CHK1, and BRCA1, as well as the level of RAD51, but not BRCA2 or SMC-1. These findings suggest that the DDR crosslinks with the immune response and both responses could be important for HPV viral replication.

Fig. 3.

STAT-5-dependent activation of ATM and ATR pathways, required for HPV genome amplification

High-risk HPV activates STAT-5 to mediate TopBP1 transcriptionally to promote ATR pathway. Meanwhile, STAT-5 does not directly regulate Tip60; instead, it partially work through PPARg to manipulate Tip60 activation which consequently facilitates ATM activation. HPV E7 oncogene is responsible for STAT-5 activation as well as interaction with other factors such as Rb, ATM, NBS1, HDAC, and p21. E6 gene may collaborate with other viral proteins to act on Tip60 in addition to its role in p53 degradation. E2 is capable of interacting with TopBP1 to mediate HPV initial replication

As a transcription factor, STAT-5 does not directly mediate the level of ATM or ATR. Instead, it deregulates the acetyltransferase Tip60 (Hong et al., 2015a) or TopBP1 (Hong et al., 2015b) to promote the activation of ATM or ATR signaling. Tip60 knockdown blocks ATM activation and HPV genome amplification upon differentiation. Both p53 and histone H2AX, the downstream targets of Tip60, may also play a role in the DDR in HPV-positive cells. Inhibition of STAT-5 activation blocks Tip60 activation and this regulation might be mediated by the kinase glycogen synthase kinase 3β (GSK3β). Consistently, GSK3β inhibition also leads to reduced levels of viral episomes and impaired genome amplification.

STAT-5 regulates transcription of the TopBP1 gene to mediate ATR activation in HPV-positive cells and knockdown of TopBP1 blocks HPV genome amplification (Hong et al., 2015b). Knockdown of TopBP1 moderately increases short-term replication of transiently transfected HPV31 plasmids and has a modest effect on stable maintenance of HPV31 episomes (Kanginakudru et al., 2015). Hong et al. (2015b) identified a critical role for TopBP1 in the differentiation-dependent late phase of the viral life cycle. TopBP1 is also recruited to viral genomes by forming complexes with the HPV E2 protein. The failure of E2 to bind TopBP1 results in a reduced replication ability, indicating that it is a positive regulator of viral replication (Donaldson et al., 2012). TopBP1 also helps to load replication factors onto origins (Gauson et al., 2015) and acts as a transcriptional regulator (Liu et al., 2003), but it is unclear if these functions are important for HPV genome amplification.

6. Perspectives

The complexity of the role of the DDR in the viral life cycle has been a recent focus of research. In the HPV field, although there have been some studies demonstrating the necessary roles of ATM or ATR signaling in HPV viral replication, more effort should be placed on the roles of the DDR factors in the HPV life cycle. What are the substrates for ATM or ATR that contribute to the HPV life cycle What other DNA damage factors are important in HPV viral replication Are DNA damage repair foci the same as HPV replication centers An additional question raised here is whether the DDR is important for the development of cervical cancer. Moreover, I presented evidence that the immune regulator STAT-5 participates in activation of the ATM or ATR pathway to facilitate HPV genome amplification, suggesting that the interaction between the innate immune response and the DNA damage pathway needs to be dissected in detail. The role of other members of STATs, such as STAT-3, in DNA damage regulation and HPV genome amplification is still not clear. Further, is the DDR able to mediate the innate immune response as a feedback loop for HPV persistent infection Resolving these questions will improve our understanding of the replication mechanisms of HPVs and provide insight for developing new therapeutic targets against HPV-related diseases.