Microhomology-mediated repair machinery and its relationship with HPV-mediated oncogenesis

Abstract

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it’s now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.

Introduction:

Globally, at least eleven percent of cancer cases are due to virus infection.^1–5 DNA viruses, such as EBV, HPV, and HBV, are some of the most commonly associated with cancer and are frequently reported to be integrated into the human genome. Integration is not a normal part of the virus lifecycle resulting in a replicatively dead virus and is considered a crucial step in carcinogenesis in nearly 15 different cancers.^6–9 Recently it has emerged that short sequences of homology are commonly found at the integration junctions of these DNA viruses and may represent an important and conserved mechanism resulting in DNA virus integration across eukaryotes. Here we review the latest evidence for the involvement of microhomology-mediated repair mechanisms in DNA virus integration during infection and tumorigenesis with a focus on HPV.

Overview of HPV-associated carcinogenesis:

HPVs belong to a heterogeneous group of non-enveloped dsDNA viruses and infect the cutaneous and mucosal squamous epithelia. Over 400 types have been sequenced, with nearly 70 types belonging to the genus Alphapapillomavirus. This genus harbors the types that can cause childhood warts, benign genital warts (low-risk HPV), or cancers of the cervix, oropharynx, vulva, anus, and penis (high-risk HPV).^10–12 These viruses depend on host DNA damage response signaling and DNA replication machinery for productive infection. Persistent HPV infection can result in carcinogenesis through sustained expression of viral oncogenes (E6, E7, E5), loss of late capsid proteins (L1, L2) and those involved in genome maintenance (E1, E2, E4) resulting in a replicatively dead virus.^13–16 Integration of the HPV genome into the host cell is frequently a crucial step in the development of cervical cancer, with HPV18 being particularly prone to integration.¹⁷ While integration is common, large sequencing studies of HPV-associated cancers have identified tumors with various combinations of episomal, integrated, and hybrid HPV-host episomes. ¹⁸ HPV was first found to be integrated in SiHa cells between the genes, KLF5 and KLF12,¹⁹ and has been detected subsequently numerous times through host-viral DNA ^19–23 or RNA ²⁴ junctions. HPV16 and HPV18, are the most abundant types found in cervical cancer worldwide. The oncogenic proteins E6 and E7 of high-risk HPVs (HR-HPVs) play pivotal roles in this process.^{12, 25, 26} One crucial interaction involves HPV16 E7 forming a complex with cellular pocket proteins (pRb, p107, and p130), which regulates cell cycle division. Furthermore, in quiescent cells, pocket proteins prohibit the activity of transcription factors like E2Fs. During cell proliferation, cyclin-dependent kinases (CDK) phosphorylate pRb, which releases active E2Fs. Binding of HPV16 E7 to the hypo-phosphorylated pRb leads to its degradation via the ubiquitin-proteasome pathway and forces cells into the S phase. Thus, the E7/pRb interaction imitates CDK-mediated phosphorylation, allowing uncontrolled cell growth.²⁷

Microhomology-mediated DNA repair machinery and Cancer:

Cells have evolved multiple strategies to sense and repair DNA double-stranded breaks (DSBs) caused by various sources of damage. There are three primary mechanisms for DSB repair—homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and microhomology-mediated end joining (MMEJ, alt-NHEJ, or alt-EJ) (Figure 1A, B, C, and D, respectively).²⁸ While HR (Figure 1B) is considered error-free, both C-NHEJ and MMEJ can generate alterations, such as insertions and deletions, around the DSB accelerating potentially carcinogenic mutagenesis.²⁸ C-NHEJ involves the direct ligation of blunt DNA ends, whereas, MMEJ is dependent on microhomologous sequences (MHS) usually less than 10 base pairs at sites of repair (Figure 1C).

Figure 1: Schematic of the mechanisms of microhomology-mediated integration of the HPV genome into the human genome.

A. Generation of concatemerized HPV genome by rolling circle amplification followed by recombination and double-strand break in the host genome. Repair of host DNA double-strand break by B. Homologous recombination (HR), or by templating with viral DNA using C. Classical non-homologous end-joining (C-NHEJ), D. Microhomology-mediated end-joining (MMEJ), E. Fork stalling and template switching (FoSTeS), or F. Microhomology-mediated break-induced replication (MMBIR). G. Circular virus-host DNA potentially produced by FoSTeS or MMEJ. H. Linear head-to-tail amplification of host and viral DNA commonly observed in HPV-associated cancers. I. Highly duplicated and rearranged HPV integration event created through repeated cycles of recombination, excision of circular DNA, amplification, and reintegration known as heterocateny. HPV DNA is shown in red, host DNA is in blue, regions of microhomologous sequence are shown in gold, recombination junctions are vertical black lines. Direction of DNA synthesis is shown by arrows with dotted lines.

C-NHEJ is dependent on the Ku70/80 heterodimer recognizing and protecting the DSB ends. Ku70/80 then interacts with DNA-dependent protein kinase catalytic subunit to control the broken end interactions. Ultimately, with C-NHEJ repair the ends are ligated together with the LIG4, XRCC4, XLF complex. Whereas MMEJ, similar to HR, acts on resected DNA ends. HR occurs during G2/S phase, utilizing sister chromatids as homologous templates. DNA resection, activated by CtIP and the MRE11-RAD50-NBS1 complex, generates short single-stranded DNA (ssDNA), which is next processed for the removal of longer ssDNA by nucleases and helicases (Figure 1B and 1D).²⁹ BRCA1 and BRCA2, along with RPA and RAD51 act as mediator proteins during this process to protect the ssDNA as well as promote strand invasion and homology searching to resolve the DSB.²⁸

MMEJ, a repair pathway that exists across various organisms including humans, has been broadly studied in the past two decades. It was initially considered as a backup pathway, but mounting evidence indicates it can be a preferred repair mechanism, especially in HR-defective backgrounds.³⁰ Identifying key MMEJ factors has been challenging due to their overlapping functions with other repair pathways, making it arduous to fully understand the underlying physiological role of MMEJ. However, unraveling the molecular mechanisms of MMEJ may open new possibilities for the development of novel cancer therapeutics, particularly in HR-deficient tumors.

BRCA1 and BRCA2 both are key factors involved in facilitating HR.³¹ Germline defects in these genes are substantial risk factors for developing various cancers and these genes are commonly inactivated across tumor types. In the absence of functional HR, MMEJ can predominate. Studies have shown that BRCA1-deficient cells exhibit an increased reliance on alternative DNA repair mechanisms, including MMEJ, to manage DNA lesions.^{32, 33} BRCA1-deficient cells were found to exhibit higher levels of MMEJ activity compared to BRCA1-proficient cells, implying that MMEJ might be upregulated as an alternative repair mechanism in the absence of functional BRCA1.³⁴ Moreover, studies have highlighted a role for both BRCA1 and BRCA2 in regulating the balance between different repair pathways, including MMEJ. ^34–36

Genomic and transcriptomic studies have revealed a predisposition towards MMEJ in some cancers, such as chronic myeloid leukemia ^{37, 38} and some resistant breast cancer cell lines, ³⁹ which exhibit higher expression of LIG3 and PARP1. Genomic rearrangements corresponding with MMEJ can also be observed in various hematological and solid tumors, particularly those with deficiencies in HR. ^{37, 40} HR-deficient ovarian tumors show higher expression of polymerase theta (POLQ), and knocking down POLQ or inhibiting PARP1, which effectively kills HR-deficient cells, as both are associated with MMEJ, indicating a dependency on MMEJ for cell proliferation in the absence of HR. ^{41, 42}

Cancer genomes often harbor interchromosomal translocations some of which are key drivers of tumorigenesis, and therefore the study of end joining has attracted considerable attention.⁴³ Some tumors undergo an event call chromothripsis, initiated by genome wide DBS and subsequent repair resulting in numerous complex rearrangements in chromosomes. While conventionally linked with C-NHEJ, recent evidence has identified MHS at chromothripsis-associated and non-chromothripsis-associated junctions in tumors suggesting the involvement of other mechanisms such as MMEJ ⁴⁴. Studies using mouse cells lacking the ligase, Lig4, or Ku70, which binds DSB ends and recruits repair factors, showed an increase in translocation frequency without changes in MHS observed at the repair junctions, suggesting a role for MMEJ in translocation formation.⁴⁵ CtIP-depleted, POLQ⁻/⁻, LIG3⁻/⁻, or PARP inhibitor-treated cells showed reduced translocation frequencies and decreased MHS at the junctions, additionally supporting MMEJ as the primary mechanism.^46–48 However, translocations induced by endonucleases show a lower frequency of MHS at these junctions in human cells, validating catalysis by NHEJ rather than MMEJ, particularly without LIG4.^{49, 50}

Flap endonuclease 1 (FEN1) additionally has been found to play a significant role in MMEJ. FEN1 is involved in processing DNA intermediates during MMEJ, contributing to repair and generating genomic alterations. FEN1 is essential for MMEJ in human cells, as its deficiency leads to a reduction in MMEJ efficiency and increased reliance on other repair pathways.⁵¹ The involvement of FEN1 in MMEJ was found to be particularly significant when the DNA breaks have non-compatible overhangs or secondary structures that need processing before joining. FEN1 promotes MMEJ by processing and removing 5’-flaps during the repair process.⁵² This function of FEN1 is crucial for creating the single-stranded regions necessary for microhomology annealing and ligation during MMEJ.

Fork stalling and template switching (FoSTeS) is a DNA replication-associated mechanism that leads to genomic rearrangements by exploiting MHS⁵³ (Figure 1E). During DNA replication, replication forks can stall due to various factors such as DNA lesions or secondary structures. FoSTeS involves the dissociation of a stalled fork from its template and the subsequent switching to a nearby template, often on a different chromosome, resulting in replication-associated rearrangements. This mechanism utilizes MHS present at the boundary between the donor and recipient templates, facilitating strand annealing and DNA synthesis.⁵⁴ Alternatively, when fork collapse occurs, break-induced replication (BIR) can restart replication especially when DSB repair is not available. This mechanism tends to be accurate and requires homologous sequences longer than 50 bp.^{55, 56} When this process fails, translesion polymerases can initiate microhomology-mediated break-induced replication (MMBIR) when longer stretches of ssDNA are present (Figure 1F). ⁵⁷ These longer stretches of ssDNA can occur not only at stalled replication forks, but also at stalled transcription complexes, excision repair tracts, or in DNA secondary structures potentially leading to many initiating situations for MMBIR. This may explain why MMBIR leads to complex rearrangements compared to BIR. FoSTeS and MMBIR have been proposed as major contributors to amplifications and rearrangements in cancer. FoSTeS and MMBIR have also been implicated in gene duplication and exon shuffling that can cause inherited germline genetic diseases and disorders as well as genomic evolution. ^{54, 58} These studies collectively illustrate the mechanistic significance of fork stalling and template switching, particularly the role of microhomology, in generating genomic rearrangements.

Evidence of Microhomology-mediated DNA repair machinery in HPV integration:

Integration of HPV DNA into the host genome is frequently an initiating factor for cervical carcinogenesis, and the level of HPV-DNA integration has been reported as a marker for disease progression.^59–63 The fraction of the HPV genome integrated, and the number of viral genome copies is highly variable and is in part affected by recombination of the viral genome concatemer prior to integration (Figure 1A). Through whole genome sequencing of HPV-associated tumors and cell lines, frequent head-to-tail amplifications of host DNA flanked by viral DNA were observed, but the underlying mechanism of these structures was unknown.⁷ Data-mining of 3,667 HPV integration breakpoints from high throughput sequencing data of cervical intraepithelial neoplasias, cervical carcinomas, and cell lines identified the presence of MHS between the human and HPV genomes near integration breakpoints, especially with MHS lengths of 4 bp or more, suggesting MHS-mediated DNA repair pathways are likely the central mechanisms involved in promoting the integration process.⁶⁴ Additional studies from whole genome sequencing data also hypothesized the formation of circular virus-host DNA intermediates after MH-mediated repair that then amplified through aberrant firing of the viral origin of replication and resulting in the observed head-to-tail concatemers of virus and host DNA (Figure 1G and 1H).⁶

MMEJ may cause lethal repair events and contribute to radiosensitivity in HR-deficient breast and ovarian tumors.^{65, 66} Similarly, HPV-positive head and neck squamous cell carcinoma (HNSCC) cells exhibit higher inherent radiosensitivity compared to HPV-negative cells, pointing out a direct role of HPV infection in radiosensitivity.^{67, 68} This, in turn, supports MMEJ activation in HPV-positive cancers. A study with 32 different cancer types, evaluated individual deletion breakpoints identified by the TCGA project and found that ovarian cancers, which often have HR defects, had the highest proportion of MHS-associated deletions. On the other hand, HNSCCs overall showed an intermediate amount of MHS, and HPV-positive HNSCCs exhibited a higher proportion of deletions associated with 3+ bp of MHS, indicating enhanced utilization of MMEJ in these cases without the typical genomic characteristics of HR deficiency.⁶⁹ Leeman et al., also reported that E7 protein significantly increased radiosensitivity by suppressing NHEJ events by 30–50%. This led to a shift towards error-prone MMEJ and HR pathways, which could explain the radiosensitivity of HPV-positive squamous cell carcinomas. Moreover, the existence of HPV in HNSCC genomes leads to an increased occurrence of MH at deletion sites, suggesting a higher propensity on the MMEJ pathway compared to HPV-negative HNSCC.⁶⁹

A recent study using an HPV integration site capture technique, also validated that MH-mediated repair is the prime mechanism behind HPV integration. Indeed, the exact locations of HPV16 integration sites were found within open chromatin areas of their integrant clones, illustrating that MH-mediated repair machineries are likely responsible for the occurrence of both ‘direct’ and ‘looping’ HPV-integration.⁷⁰ Another study using long-read sequencing of HPV-positive oropharyngeal cancers and human cell lines identified a new form of genomic structural variation mediated by recurrent recombination, excision, and amplification of HPV and host DNA called ‘heterocateny’ (Figure 1I).⁷¹ While this study did not evaluate the involvement of MMEJ, based on the findings of other studies suggest that this may be part of the underlying mechanism and is worth investigating further.

Mechanisms of microhomology-mediated DNA repair machinery in HPV integration

As discussed above, substantial data suggest the involvement of MMEJ in HPV integration. However, the important question will be the mechanism of connecting the breakage of the human genome through MHS-mediated DNA repair pathways in HPV integration. Integration of HPV into the introns of FHIT and LRP1B led to downregulation of protein expression, while integration into flanking regions resulted in elevated expression of MYC and HMGA2 proteins.⁶⁴ In addition, MHS between human and HPV genomes were found to be significantly enriched near integration breakpoints. Therefore, a novel viral integration model was proposed, suggesting that during HPV infection unstable local genomic elements can lead to DNA breaks or stalled replication forks, activating the FoSTeS or MMBIR pathways, and potentially facilitating HPV’s fusion to the host genome through microhomology-mediated DNA repair pathways to complete the integration process.⁶⁴

Moreover, defects in C-NHEJ are linked to radiosensitivity among certain cancers.⁶⁹ The HPV E7 protein targets RB1, involved in C-NHEJ, and potentially hinders DSB repair.⁶⁹ Existing evidence showed that the influence of the E7 protein on repair machinery choices is assigned by a specific residue within its LXCXE domain, which is crucial for binding to Rb, and the E7 C24G mutant.⁷² Disruption of C-NHEJ can increase MMEJ, which is linked with HPV integration sites augmented in MHS, suggesting an HPV-MMEJ association.⁶⁹ In this study, Leeman et al. employed TCGA RNA sequencing datasets to reveal expression levels of MMEJ factors, specifically POLQ, in HPV-positive HNSCCs with strong expression of these MMEJ components including POLQ; this strong expression suggests MMEJ may indeed be an active mechanism in these instances. Collectively, this insinuates that the high MHS phenotype observed in HPV-positive HNSCCs could be correlated with enhanced utilization of MMEJ for DNA repair.⁶⁹ The viral genome, when linearized, can inadvertently integrate into the damaged host genome through the MMEJ-mediated DNA repair pathway, which is primarily executed by these five steps: end resection, annealing of the MHS, removal of the flap, fill-in synthesis, and ligation.²⁸ The first step in MMEJ involves limited resection of DSBs by host nucleases, revealing MHS.^{28, 73} CtIP and FEN1, host nucleases, give rise to a 3’ overhang necessary for POLQ-mediated MMEJ.^74–79 To activate CtIP, it needs phosphorylation at specific sites by host serine/threonine protein kinases (such as CDK1, Aurora A, and PLK1).^{35, 76, 80}

Viral genome copy number and viral oncogene expression can be highly variable in tumors, stipulating other genomic factors, such as flanking host genes, act as selective pressures for integration.⁸¹ One feature that contributes to hotspots of HPV DNA integration are common and cell-type specific chromosomal fragile sites, which can develop DBS during replication stress which also can lead to alteration in gene expression levels.^{20, 82–84} Recent studies have also found that HPV integration can occur at DBS associated with chromothripsis, which as previously mentioned can generate genome-wide chromosomal changes and have MHS at repair junctions.^{85, 86, 87, 88} Integration next to coding regions or even distant flanking regions of genes can lead to changes like amplification, rearrangement, translocation, or long-range cis interactions. This further suggests that chromatin interactions may play a vital role in the selection and development of cervical cancer.^{7, 64, 89, 90} In a study, researchers used five HPV16 integrant cloned cell lines⁸³ to explore the mechanisms behind HPV DNA integration and host gene expression modulation that correspond with selection during cancer development and progression. A novel technique called HPV integrated site capture (HISC) was developed to estimate the HPV16 integration sites throughout the genome at a nucleotide level. By combining this technique with HPV16-specific Region Capture Hi-C, researchers pinpointed integration sites coexisting with open chromatin regions and uncovered that integration likely occurs through MHS-mediated repair mechanisms. Moreover, the evidence of both direct integrations was also found, implying deletion of regions in the host genome, and a ‘looping’ mechanism, where flanking regions of the host genome become amplified.⁷⁰ Additionally, this study demonstrates how interactions between host chromatin and integrated virus genome at short and long range can greatly contribute to dysregulation of host genes as well as disordered topologically associating domains (TADs) interactions during early stages of cervical neoplasia progression.⁷⁰

Non-alpha papillomaviruses and microhomology-mediated DNA repair machinery

Beyond Alphapapillomaviruses, there are four additional genera of human papillomaviruses—Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, and Nupapillomavirus.^{91, 92} In recent years due to decreasing costs and advances in sequencing technologies, the number of known types in the genera Gamma and Beta have dramatically increased and now Gammapapillomaviruses represent the largest and most diverse genus of HPVs.⁹³ The association of these viruses with cancer is much less understood compared to Alphapapillomaviruses since these viruses have not been found to be clonally integrated in human cancers. However, similar to known oncogenic HPVs, Betapapillomavirus and Gammapapillomavirus types are dependent on host DNA repair machinery for replication and the E6 and E7 of these viruses interact with a number of host factors associated with oncogenic reprogramming.^{94, 95}

Betapapillomaviruses primarily infect cutaneous skin, and while many cause benign warts or lesions, certain Betapapillomavirus types have been implicated in the pathogenesis of non-melanoma skin cancers in immunosuppressed individuals.⁹⁶ Specific Betapapillomavirus types, such as HPV5 and HPV8, are more frequently found in the skin lesions of individuals with a rare genetic disorder, epidermodysplasia verruciformis (EV), which predisposes them to squamous cell carcinoma development.⁹⁷ The Betapapillomavirus types found in EV patients have been shown to have transforming capabilities, potentially contributing to the development of skin cancer. Moreover, studies have revealed a higher prevalence of Betapapillomavirus DNA in skin cancer lesions of immunosuppressed organ transplant recipients, suggesting a link between immunosuppression and Betapapillomavirus-associated skin cancer.^98–100 However, as these viruses are not found to be clonally integrated into the tumor genome, the carcinogenic mechanisms are unclear, but may be related to disruption of DNA repair used for virus replication.

Betapapillomavirus infection have been found to disrupt the two primary DSB repair pathways i.e., HR and C-NHEJ.¹⁰¹ However, it was found that HPV E6 proteins, specifically HPV5, 8, and 38, decrease HR efficiency. Mechanistically, they downregulate the transcription regulator p300, resulting in decreased expression of BRCA1, BRCA2, and ATM, as well as reduced the formation of BRCA1 and BRCA2 repair complexes. Although RAD51 foci formation is unaltered, the resolution of RAD51 foci is also delayed, suggesting that the repair complexes are non-functional. C-NHEJ is also hindered by HPV8 E6 by reducing the phosphorylation of DNA-PKCs and its downstream target Artemis, impairing the resolution of pDNA-PKCs complexes. These effects are closely linked with HPV8 E6-dependant destabilization of p300, while the formation of XRCC4 foci is decreased independently of p300 destabilization.¹⁰¹ By utilizing small molecule inhibitors, CRISPR/CAS9 gene knockout, and an HPV8 E6 mutant as model systems, researchers discovered that HPV8 E6 fosters MMEJ through its interaction with p300. As part of its repair pathway, MMEJ employs POLQ end joining. Furthermore, whole genome sequencing data revealed that HPV8 E6 promotes an increased frequency of deletions characterized by MHS associated with MMEJ. ¹⁰² Independent of integration, as mentioned previously, upregulation of MMEJ can lead to chromosomal translocations, copy number changes, and even small indels contributing to carcinogenesis. In combination with HPV sequestering host DNA replication machinery in combination with UV irradiation may cause extensive damage.

Mouse papillomavirus (MmuPV1), belonging to the genus Pipapillomavirus, has recently emerged as an important in vivo model for HPV-mediated carcinogenesis.^103–105 Studies using this model system further supported the involvement of MMEJ-mediated DNA joining in PV integration.¹⁰⁶ Yu and colleagues identified MHS of 2–10 nucleotides at the integration junction regions, as well as elevated expression of DNA repair and MMEJ factors, (i.e., H2ax, Fen1, Polq, Cdk1, and Plk1) in MmuPV1-infected tissues and tumors as compared to normal tissues. Mdc1 expression was found to be downregulated, further validating the association of MMEJ in MmuPV1 integration events.¹⁰⁶ Enhanced CtIP expression and S327 phosphorylation were also reported in MmuPV1 tumor tissues. This phosphorylation cascade triggers CtIP binding to PLK1 and facilitates further phosphorylation of CtIP at S723, initiating the end resection process.¹⁰⁶

Evidence for integration of other DNA viruses by microhomology-mediated DNA repair:

Polyomaviruses are another family of small DNA viruses related to papillomaviruses that cause ubiquitous subclinical infections in immunocompetent individuals. Merkel cell polyomavirus (MCPyV) was the first human polyomavirus to be clearly associated with carcinogenesis in humans and is the primary etiologic agent of Merkel cell carcinoma (MCC).¹⁰⁷ From three studies of primary MCC and MCC cell lines, two major general groups of integration patterns were identified. The first group exhibited simple, linear integration signatures with minimal loss or amplification of host sequences. The second group showed complex integration patterns with host genomic amplifications, which is potentially a result of MMBIR or rolling circle amplification of the viral-host circular DNA intermediate after MMEJ. In one study, significant enrichment of homologies was not found by the global alignment analysis.¹⁰⁸ However, the researchers developed a model to estimate homology scores for both sides of each virus-host junction, and it was found that in Z-pattern integration, MHS on the virus side was significantly enriched, while they had a lesser role or no role in linear integration patterns. Collectively, these findings suggest that MCPyV-integration patterns involve MHS to initiate or promote MCC.¹⁰⁸

BK polyomavirus (BKPyV) can cause nephropathy and hemorrhagic cystitis in transplant recipients. Recent data supports that this virus plays a role in bladder carcinogenesis of solid organ transplant recipients and several studies have now evaluated integration of BKPyV by long-read, whole genome sequencing, and hybrid capture.^{109, 110, 111, 112} All of these studies found enrichment of MHS at sites of BKPyV integration. One study using WGS also identified that these events correspond to amplification of flanking host DNA as has been observed in HPV and MCPyV-associated tumors. While this process and increased expression of MMEJ-associated genes, such as POLQ, have been observed in cellular models of BKPyV infection, recombination within the BKPyV genome from an in vitro model of BKPyV persistence and replication in renal proximal tubule epithelial cells did not show an enrichment of MHS.^{113, 114} This may suggest that recombination between viral genomes and the recombination that leads to integration into the host genome are mechanistically distinct. However, pathogenic rearrangements in the polyomavirus bidirectional promoter have potentially been attributed to FEN1; raising more questions about the involvement of MMEJ in viral recombination. ¹¹⁵

As has already been noted, HPV, HBV, and EBV all similarly integrate into the human genome and can cause cancer. By globally analyzing the integration junctions and patterns of tumors containing these viruses, Tian and colleagues identified that a common integration mechanism termed synthesis-dependent end-joining likely explains the multiple types of breakpoints, including those with blunt ends, short insertions, and MHS, unifying and supporting previous studies on viral DNA integration in cancer.^{9 116 64, 69} This study also identified overlapping integration loci that affect genes among those three viruses, potentially revealing even more commonalities between these viruses and their mechanisms of carcinogenesis.

Not all DNA viruses, human tropic or otherwise, follow this pattern. For example, adeno-associated virus (AAV) has also been observed to integrate with MHS (reviewed in ¹¹⁷). The wildtype virus preferentially integrates into chromosome 19 mediated by the viral replicase and contributes to viral latency; whereas, the derived vectors missing this gene integrate at a lower rate in unstable regions of the host genome. Lastly, other more divergent DNA viruses of invertebrates have been observed to integrate through MHS-related mechanisms as evidenced by the symbiotic polydnaviruses of parasitoid wasps.^{118, 119} None of these viruses are associated with cancers of their respective hosts, and integration seems to be part of the virus lifecycle, unlike the previously discussed viruses. Additionally, the exact type of MHS-mediated repair pathway (MMEJ, FoSTeS, MMBIR) involved is unknown in these cases. This, plus the observation that many endogenized viruses also show evidence of MHS-mediated integration, raises the question of whether MHS-mediated repair pathways are directly beneficial for the virus or serve as an innate immune response for the host. In mammals, DNA sensors are intimately entwined with immune signaling pathways (reviewed in ¹²¹). Cytoplasmic DNA sensors, such as cGAS-STING, are the first line to detect misplaced DNA such as through infection or extensive DNA damage. However, to-date MHS-mediated repair pathways have not been linked to immune signaling. Further studies are needed to fully delineate the roles of MHS-mediated repair pathways in virus infections.

Conclusions:

MHS are commonly observed at the sites of integration for HPV in tumor genomes. Erroneous repair pathways that utilize MHS, such as MMEJ, MMBIR, and FoSTeS, have all been implicated in genome instability, translocations, and gene duplications that can lead to cancer and germline diseases and disorders. Genes and their products associated with these pathways are upregulated during papillomavirus infection in humans and mice. Additionally, papillomavirus proteins have been shown to directly antagonize C-NHEJ and HR pathways, leading to increasing MMEJ usage in infected cells. The manipulation of these pathways by papillomaviruses is likely in direct service of using the host machinery to successfully replicate its own dsDNA genome. MMEJ upregulation in combination with this sequestering of host replication machinery likely enhances the unintended byproducts of virus integration and host mutagenesis. Other DNA viruses, including polyomaviruses, HBV, EBV, and polydnaviruses of invertebrates all have been observed to integrate commonly flanked by MHS showing this to be a general and evolutionarily old mechanism. However, there is conflicting data as to why this is common. For some of the discussed viruses, integration is part of their lifecycle, and this does not appear to cause malignancy in the host, so MHS-mediated integration is beneficial. However, for the viruses where integration is a dead-end for replication and leads to cancer or endogenization MHS-mediated integration massively disrupts the normal route for virus survival suggesting that this may be a type of innate immune response for the host. This paradox highlights the complexity of these processes and the continued need for research in these fields.

Data availability statement:

No data were generated for this study.