Beta Human Papillomavirus 8 E6 Induces Micronucleus Formation and Promotes Chromothripsis.

Cutaneous beta genus human papillomaviruses (ß-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of ß-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from ß-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (ß-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from ß-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

The potential of PCNA inhibition as a therapeutic strategy in cervical cancer.

Cervical cancers are the fourth most common and most deadly cancer in women worldwide. Despite being a tremendous public health burden, few novel approaches to improve care for these malignancies have been introduced. We discuss the potential for proliferating cell nuclear antigen (PCNA) inhibition to address this need as well as the advantages and disadvantages for compounds that can therapeutically inhibit PCNA with a specific focus on cervical cancer.

Predicting the Prognostic Value of POLI Expression in Different Cancers via a Machine Learning Approach.

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases (POLI) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival (p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival.

Beta Human Papillomavirus 8E6 Attenuates LATS Phosphorylation after Failed Cytokinesis.

Beta genus human papillomaviruses (ß-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, ß-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, ß-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how ß-HPV E6 causes this change in cell signaling. An in silico screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. ß-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that ß-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on ß-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. ß-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a ß-HPV E6 protein has been shown to hinder HP signaling.IMPORTANCE ß-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of ß-HPV biology is warranted. If ß-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome. In vitro data support this idea by demonstrating the ability of the ß-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that ß-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, ß-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, ß-HPV 8E6 does not limit senescence associated with failed cytokinesis.

High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway.

Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA.IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes.

ß-HPV 5 and 8 E6 disrupt homology dependent double strand break repair by attenuating BRCA1 and BRCA2 expression and foci formation.

Recent work has explored a putative role for the E6 protein from some ß-human papillomavirus genus (ß-HPVs) in the development of non-melanoma skin cancers, specifically ß-HPV 5 and 8 E6. Because these viruses are not required for tumor maintenance, they are hypothesized to act as co-factors that enhance the mutagenic capacity of UV-exposure by disrupting the repair of the resulting DNA damage. Supporting this proposal, we have previously demonstrated that UV damage signaling is hindered by ß-HPV 5 and 8 E6 resulting in an increase in both thymine dimers and UV-induced double strand breaks (DSBs). Here we show that ß-HPV 5 and 8 E6 further disrupt the repair of these DSBs and provide a mechanism for this attenuation. By binding and destabilizing a histone acetyltransferase, p300, ß-HPV 5 and 8 E6 reduce the enrichment of the transcription factor at the promoter of two genes critical to the homology dependent repair of DSBs (BRCA1 and BRCA2). The resulting diminished BRCA1/2 transcription not only leads to lower protein levels but also curtails the ability of these proteins to form repair foci at DSBs. Using a GFP-based reporter, we confirm that this reduced foci formation leads to significantly diminished homology dependent repair of DSBs. By deleting the p300 binding domain of ß-HPV 8 E6, we demonstrate that the loss of robust repair is dependent on viral-mediated degradation of p300 and confirm this observation using a combination of p300 mutants that are ß-HPV 8 E6 destabilization resistant and p300 knock-out cells. In conclusion, this work establishes an expanded ability of ß-HPV 5 and 8 E6 to attenuate UV damage repair, thus adding further support to the hypothesis that ß-HPV infections play a role in skin cancer development by increasing the oncogenic potential of UV exposure.

Manipulation of cellular DNA damage repair machinery facilitates propagation of human papillomaviruses.

In general, the interplay among viruses and DNA damage repair (DDR) pathways can be divided based on whether the interaction promotes or inhibits the viral lifecycle. The propagation of human papillomaviruses is both promoted and inhibited by DDR proteins. As a result, HPV proteins both activate repair pathways, such as the ATM and ATR pathways, and inhibit other pathways, most notably the p53 signaling pathway. Indeed, the role of HPV proteins, with regard to the DDR pathways, can be divided into two broad categories. The first set of viral proteins, HPV E1 and E2 activate a DNA damage response and recruit repair proteins to viral replication centers, where these proteins are likely usurped to replicate the viral genome. Because the activation of the DDR response typically elicits a cell cycle arrest that would impeded the viral lifecycle, the second set of HPV proteins, HPV E6 and E7, prevents the DDR response from pausing cell cycle progression or inducing apoptosis. This review provides a detailed account of the interactions among HPV proteins and DDR proteins that facilitate HPV propagation.

Beta human papillomavirus E6 expression inhibits stabilization of p53 and increases tolerance of genomic instability.

UNLABELLED: Infections with the beta genus of human papillomaviruses (ß-HPVs) may contribute to the development of nonmelanoma skin cancers. However, ß-HPV genomes are found at too low a copy number in tumors for the virus to be necessary for tumor maintenance. Instead, they are hypothesized to destabilize the host genome by allowing the persistence of mutations that can drive tumorigenesis independently of the viral genome. Supporting this premise is our previous finding that the expression of some ß-HPV E6 proteins can attenuate p53 signaling in response to DNA damage. We show that ß-HPV E6 proteins can prevent the stabilization of p53 in response to two types of genome-destabilizing events, aberrant mitosis and dysregulated centrosome duplication. The inability to stabilize p53 in response to these stimuli allows cells expressing HPV5, HPV8, or HPV38 E6 to remain proliferatively active, leading to further genome deterioration in a proportion of these cells. These phenotypes are lost by the introduction of a mutation into the p300 binding domain of HPV8 E6 or by the transfection of mutated p300 that is resistant to the degradation mediated by HPV5 or HPV8 E6. These findings expand the understanding of the role played by p300 in promoting the faithful resolution of mitotic figures as well as proper centrosome duplication. Finally, we describe a phenomenon by which binucleated cells are resolved via cytokinesis into two cells, each with one nucleus. These data support the hypothesis that ß-HPV infections may promote tumorigenesis via genome destabilization. IMPORTANCE: The work described in this report provides support for the hypothesis that ß-HPV infections may contribute to nonmelanoma skin cancer by increasing the likelihood that tumorigenic mutations are introduced into the host cell's genome. We demonstrate that expression of the E6 proteins from some of these viruses increases the tolerance of two genome-destabilizing events, aberrant cell division and dysregulated centrosome duplication. Typically, these mutagenic occurrences elicit the stabilization of the tumor suppressor p53, which prevents further propagation of cells containing these errors. We show that the expression of ß-HPV E6 restricts this stabilization of p53, leading not only to continued cellular proliferation but also to further accumulation of similar mutagenic events. Finally, in addition to supporting a role for ß-HPV infections in certain skin cancers, we present studies with a mutated ß-HPV E6 protein suggesting that the histone acetyltransferase p300 plays a role in promoting genome stability during replication.

HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage.

The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the ß human papillomavirus genus (ß-HPVs). Some ß-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain ß-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that ß-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.

Interactions among human papillomavirus proteins and host DNA repair factors differ during the viral life cycle and virus-induced tumorigenesis.

This review focuses on the impact of human papillomavirus (HPV) oncogenes on DNA repair pathways with a particular focus on how these relationships change as productive HPV infections transition to malignant lesions. We made specific efforts to incorporate advances in the understanding of HPV and DNA damage repair over the last 4 years. We apologize for any articles that we missed in compiling this report.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation.

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other ß-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants.

Cervical Cancer Development: Implications of HPV16 E6E7-NFX1-123 Regulated Genes.

High-risk human papillomavirus (HR HPV) causes nearly all cervical cancers, half of which are due to HPV type 16 (HPV16). HPV16 oncoprotein E6 (16E6) binds to NFX1-123, and dysregulates gene expression, but their clinical implications are unknown. Additionally, HPV16 E7's role has not been studied in concert with NFX1-123 and 16E6. HR HPVs express both oncogenes, and transformation requires their expression, so we sought to investigate the effect of E7 on gene expression. This study's goal was to define gene expression profiles across cervical precancer and cancer stages, identify genes correlating with disease progression, assess patient survival, and validate findings in cell models. We analyzed NCBI GEO datasets containing transcriptomic data linked with cervical cancer stage and utilized LASSO analysis to identify cancer-driving genes. Keratinocytes expressing 16E6 and 16E7 (16E6E7) and exogenous NFX1-123 were tested for LASSO-identified gene expression. Ten out of nineteen genes correlated with disease progression, including CEBPD, NOTCH1, and KRT16, and affected survival. 16E6E7 in keratinocytes increased CEBPD, KRT16, and SLPI, and decreased NOTCH1. Exogenous NFX1-123 in 16E6E7 keratinocytes resulted in significantly increased CEBPD and NOTCH1, and reduced SLPI. This work demonstrates the clinical relevance of CEBPD, NOTCH1, KRT16, and SLPI, and shows the regulatory effects of 16E6E7 and NFX1-123.

Catching HPV in the Homologous Recombination Cookie Jar.

To replicate, the human papillomaviruses (HPVs) that cause anogenital and oropharyngeal malignancies must simultaneously activate DNA repair pathways and avoid the cell cycle arrest that normally accompanies DNA repair. For years it seemed that HPV oncogenes activated the homologous recombination pathway to facilitate the HPV lifecycle. However, recent developments show that, although homologous recombination gene expression and markers of pathway activation are increased, homologous recombination itself is attenuated. This review provides an overview of the diverse ways that HPV oncogenes manipulate homologous recombination and ideas on how the resulting dysregulation and inhibition offer opportunities for improved therapies and biomarkers.

ß-HPV 8E6 combined with TERT expression promotes long-term proliferation and genome instability after cytokinesis failure.

Human papillomavirus (HPV) is a family of viruses divided into five genera: alpha, beta, gamma, mu, and nu. There is an ongoing discussion about whether beta genus HPVs (ß-HPVs) contribute to cutaneous squamous cell carcinoma (cSCC). The data presented here add to this conversation by determining how a ß-HPV E6 protein (ß-HPV 8E6) alters the cellular response to cytokinesis failure. Specifically, cells were observed after cytokinesis failure was induced by dihydrocytochalasin B (H2CB). ß-HPV 8E6 attenuated the immediate toxicity associated with H2CB but did not promote long-term proliferation after H2CB. Immortalization by telomerase reverse transcriptase (TERT) activation also rarely allowed cells to sustain proliferation after H2CB exposure. In contrast, TERT expression combined with ß-HPV 8E6 expression allowed cells to proliferate for months following cytokinesis failure. However, this continued proliferation comes with genome destabilizing consequences. Cells that survived H2CB-induced cytokinesis failure suffered from changes in ploidy.

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity.

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (ß-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that ß-HPV promotes tumorigenesis, while others suggest that ß-HPV protects against skin cancer. Most of the molecular evidence that ß-HPV impairs DNA repair has been gained via characterization of the E6 protein from ß-HPV 8 (ß-HPV 8E6). Moreover, ß-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, ß-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, ß-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, ß-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, ß-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of ß-HPV 8E6.

DNA repair gene expression is increased in HPV positive head and neck squamous cell carcinomas.

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.

High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction.

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

mSphere of Influence: the Value of Simplicity in Experiments and Solidarity among Lab Members.

Nicholas Wallace studies how human papillomaviruses cause cancer throughout the genital and oropharyngeal tracts as well as in the skin. These viruses inhibit host DNA repair to promote their life style and in doing increase the risk of oncogenic mutations. In this mSphere of Influence article, he reflects on how two papers influenced him. "Human Papillomaviruses Activate the ATM DNA Damage Pathway for Viral Genome Amplification upon Differentiation" by C. A. Moody and L. Laimins (PLoS Pathog 5:e10000605, 2009, https://doi.org/10.1371/journal.ppat.1000605) reminded him of the power of straightforward approaches, while "Forty-Five Years of Cell-Cycle Genetics" by B. Reid et al. (B. J. Reid, J. G. Culotti, R. S. Nash, and J. R. Pringle, Mol Biol Cell 26:4307-4312, 2015, https://doi.org/10.1091/mbc.E14-10-1484) gave him the inspiration for his lab management style.