Crowd Control: E7 Conservation Is the Key to Cancer.

Several human papillomavirus type 16 (HPV16) oncoproteins contribute to cellular transformation in vitro. In this issue of Cell, Mirabello and colleagues use high-throughput sequencing data to assess the diversity of HPV16 isolates from human patients. These data suggest that the E7 oncoprotein is the fundamental contributor to in vivo carcinogenesis.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

MmuPV1 E7's interaction with PTPN14 delays Epithelial differentiation and contributes to virus-induced skin disease.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.

Human papillomaviruses: Knowns, mysteries, and unchartered territories.

There has been an explosion in the number of papillomaviruses that have been identified and fully sequenced. Yet only a minute fraction of these has been studied in any detail. Most of our molecular research efforts have focused on the E6 and E7 proteins of "high-risk," cancer-associated human papillomaviruses (HPVs). Interactions of the high-risk HPV E6 and E7 proteins with their respective cellular targets, the p53 and the retinoblastoma tumor suppressors, have been investigated in minute detail. Some have thus questioned if research on papillomaviruses remains an exciting and worthwhile area of investigation. However, fundamentally new insights on the biological activities and cellular targets of the high-risk HPV E6 and E7 proteins have been discovered and previously unstudied HPVs have been newly associated with human diseases. HPV infections continue to be an important cause of human morbidity and mortality and since there are no antivirals to combat HPV infections, research on HPVs should remain attractive to new investigators and biomedical funding agencies, alike.

PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis.

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion of PTPN14 from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation and PTPN14 deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+ but not HPV- cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

KDM6A-Mediated Expression of the Long Noncoding RNA DINO Causes TP53 Tumor Suppressor Stabilization in Human Papillomavirus 16 E7-Expressing Cells.

Human papillomavirus 16 (HPV16) E7 has long been known to stabilize the tumor suppressor TP53. However, the molecular mechanism of TP53 stabilization by HPV16 E7 has remained obscure, and this stabilization can occur independently of the E2F-regulated MDM2 inhibitor p14ARF Here, we report that the damage-induced noncoding (DINO) lncRNA (DINOL) is the "missing link" between HPV16 E7 and increased TP53 levels. DINO levels are decreased in cells where TP53 is inactivated, either by HPV16 E6, by expression of a dominant negative TP53 minigene, or by TP53 depletion. DINO levels are increased in HPV16 E7-expressing cells. HPV16 E7 causes increased DINO expression independently of RB1 degradation and E2F1 activation. Similar to what is seen with the adjacent CDKN1A locus, DINO expression is regulated by the histone demethylase KDM6A. DINO stabilizes TP53 in HPV16 E7-expressing cells, and as it is a TP53 transcriptional target, DINO levels further increase. As with expression of other oncogenes, such as adenovirus E1A or MYC, HPV16 E7-expressing cells are sensitized to cell death under conditions of metabolic stress, which in the case of E7 has been linked to TP53 activation. Consistent with earlier studies, we show that HPV16 E7-expressing keratinocytes are highly sensitive to metabolic stress induced by starvation or the antidiabetic drug metformin. Sensitivity of HPV16 E7-expressing cells to metabolic stress is rescued by DINO depletion. Moreover, DINO depletion decreases sensitivity to the DNA damage-inducing chemotherapy agent doxorubicin. This work identifies DINO as a critical mediator of TP53 stabilization and activation in HPV16 E7-expressing cells.IMPORTANCE Viral oncoproteins, including HPV16 E6 and E7, have been instrumental in elucidating the activities of cellular signaling networks, including those governed by the TP53 tumor suppressor. Our study demonstrates that the long noncoding RNA DINO is the long-sought missing link between HPV16 E7 and elevated TP53 levels. Importantly, the TP53-stabilizing DINO plays a critical role in the cell death response of HPV16 E7-expressing cells to metabolic stress or DNA damage.

A Conserved Amino Acid in the C Terminus of Human Papillomavirus E7 Mediates Binding to PTPN14 and Repression of Epithelial Differentiation.

The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPVs bind to the host cellular nonreceptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Here, we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates the interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes, and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now, we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the life span of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte life span extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity.IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment, we demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte life span. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated, and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.

The Human Papillomavirus E6 Oncoprotein Targets USP15 and TRIM25 To Suppress RIG-I-Mediated Innate Immune Signaling.

Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-ß, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.

Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-ß Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation.

Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-ß signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.

KDM6A addiction of cervical carcinoma cell lines is triggered by E7 and mediated by p21CIP1 suppression of replication stress.

Expression of E7 proteins encoded by carcinogenic, high-risk human papillomaviruses (HPVs) triggers increased expression of the histone H3 lysine 27 demethylase KDM6A. KDM6A expression is necessary for survival of high-risk HPV E7 expressing cells, including several cervical cancer lines. Here we show that increased KDM6A in response to high-risk HPV E7 expression causes epigenetic de-repression of the cell cycle and DNA replication inhibitor p21CIP1, and p21CIP1 expression is necessary for survival of high-risk HPV E7 expressing cells. The requirement for KDM6A and p21CIP1 expression for survival of high-risk HPV E7 expressing cells is based on p21CIP1's ability to inhibit DNA replication through PCNA binding. We show that ectopic expression of cellular replication factors can rescue the loss of cell viability in response to p21CIP1 and KDM6A depletion. Moreover, we discovered that nucleoside supplementation will override the loss of cell viability in response to p21CIP1 depletion, suggesting that p21CIP1 depletion causes lethal replication stress. This model is further supported by increased double strand DNA breaks upon KDM6A or p21CIP1 depletion and DNA combing experiments that show aberrant re-replication upon KDM6A or p21CIP1 depletion in high-risk HPV E7 expressing cells. Therefore, KDM6A and p21CIP1 expression are essential to curb E7 induced replication stress to levels that do not markedly interfere with cell viability.

Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.

A Mathematical Model of Cell Cycle Dysregulation Due to Human Papillomavirus Infection.

Human papillomaviruses (HPVs) that infect mucosal epithelium can be classified as high risk or low risk based on their propensity to cause lesions that can undergo malignant progression. HPVs produce the E7 protein that binds to cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein (RB) to modulate cell cycle control. Generally, high-risk HPV E7 proteins bind to RB with a higher affinity than low-risk HPV E7s, but both are able to deactivate RB and trigger S phase progression. In uninfected cells, RB inactivation is a tightly controlled process that must coincide with growth factor stimulation to commit cells to division. High-risk HPV E7 proteins short-circuit this control by decreasing growth factor requirement for cell division. We develop a mathematical model to examine the role that RB binding affinity, growth factor concentration, and E7 concentration have on cell cycle progression. Our model predicts that high RB binding affinity and E7 concentration accelerate the [Formula: see text] to S phase transition and weaken the dependence on growth factor. This model thus captures a key step in high-risk HPV oncogenesis.

Human papillomavirus carcinogenesis: an identity crisis in the retinoblastoma tumor suppressor pathway.

Viruses are obligate intracellular parasites and need to reprogram host cells to establish long-term persistent infection and/or to produce viral progeny. Cellular changes initiated by the virus trigger cellular defense responses to cripple viral replication, and viruses have evolved countermeasures to neutralize them. Established models have suggested that human papillomaviruses target the retinoblastoma (RB1) and TP53 tumor suppressor networks to usurp cellular replication, which drives carcinogenesis. More recent studies, however, suggest that modulating the activity of the Polycomb family of transcriptional repressors and the resulting changes in epigenetic regulation are proximal steps in the rewiring of cellular signaling circuits. Consequently, RB1 inactivation evolved to tolerate the resulting cellular alterations. Therefore, epigenetic reprograming results in cellular "addictions" to pathways for survival. Inhibition of such a pathway could cause "synthetic lethality" in adapted cells while not markedly affecting normal cells and could prove to be an effective therapeutic approach.

Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines.

The tumor suppressor p16(INK4A) inhibits formation of enzymatically active complexes of cyclin-dependent kinases 4 and 6 (CDK4/6) with D-type cyclins. Oncogenic stress induces p16(INK4A) expression, which in turn triggers cellular senescence through activation of the retinoblastoma tumor suppressor. Subversion of oncogene-induced senescence is a key step during cancer development, and many tumors have lost p16(INK4A) activity by mutation or epigenetic silencing. Human papillomavirus (HPV)-associated tumors express high levels of p16(INK4A) in response to E7 oncoprotein expression. Induction of p16(INK4A) expression is not a consequence of retinoblastoma tumor suppressor inactivation but is triggered by a cellular senescence response and is mediated by epigenetic derepression through the H3K27-specific demethylase (KDM)6B. HPV E7 expression causes an acute dependence on KDM6B expression for cell survival. The p16(INK4A) tumor suppressor is a critical KDM6B downstream transcriptional target and its expression is critical for cell survival. This oncogenic p16(INK4A) activity depends on inhibition of CDK4/CDK6, suggesting that in cervical cancer cells where retinoblastoma tumor suppressor is inactivated, CDK4/CDK6 activity needs to be inhibited in order for cells to survive. Finally, we note that HPV E7 expression creates a unique cellular vulnerability to small-molecule KDM6A/B inhibitors.

Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development.

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

The HPV16 E6 oncoprotein causes prolonged receptor protein tyrosine kinase signaling and enhances internalization of phosphorylated receptor species.

The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins.

Are selective estrogen receptor modulators (SERMs) a therapeutic option for HPV-associated cervical lesions and cancers?

This Commentary highlights the article by Spurgeon et al which evaluates oral SERM raloxifene as a potential therapeutic agent for HPV-associated cancer.