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 E6 induces cell proliferation and other hallmarks of cancer.

The E6 protein encoded by the murine papillomavirus (MmuPV1) is essential for MmuPV1-induced skin disease. Our previous work has identified a number of cellular interacting partners of MmuPV1 E6 and E7 through affinity purification/mass spectrometry analysis. These studies revealed that MmuPV1 E6 potently inhibits keratinocyte differentiation through multiple molecular mechanisms including inhibition of NOTCH and TGF-ß signaling. Here, we report that MmuPV1 E6 has additional important oncogenic activities when expressed in its natural host cells, mouse keratinocytes, including increasing proliferation, overcoming density-mediated growth arrest, and proliferation under conditions of limited supply of growth factors. Unbiased proteomic/transcriptomic analyses of mouse keratinocytes expressing MmuPV1 E6 substantiated its effect on these cellular processes and divulged that some of these effects may be mediated in part through it upregulating E2F activity. Our analyses also revealed that MmuPV1 E6 may alter other cancer hallmarks including evasion of growth suppressors, inhibition of immune response, resistance to cell death, and alterations in DNA damage response. Collectively, our results suggest that MmuPV1 E6 is a major driver of multiple hallmarks of cancer in MmuPV1's natural host cells, mouse keratinocytes.IMPORTANCEThe Mus musculus papillomavirus 1 (MmuPV1) E6 and E7 proteins are required for MmuPV1-induced disease. Our understanding of the activities of MmuPV1 E6 has been based on affinity purification/mass spectrometry studies where cellular interacting partners of MmuPV1 E6 were identified, and these studies revealed that MmuPV1 E6 can inhibit keratinocyte differentiation through multiple mechanisms. We report that MmuPV1 E6 encodes additional activities including the induction of proliferation, resistance to density-mediated growth arrest, and decreased dependence on exogenous growth factors. Proteomic and transcriptomic analyses provided evidence that MmuPV1 E6 increases the expression and steady state levels of a number of cellular proteins that promote cellular proliferation and other hallmarks of cancer. These results indicate that MmuPV1 E6 is a major driver of MmuPV1-induced pathogenesis.

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.

The HPV8 E6 protein targets the Hippo and Wnt signaling pathways as part of its arsenal to restrain keratinocyte differentiation.

IMPORTANCE: Human papillomaviruses (HPVs) infect basal epithelial cells and cause a dramatic expansion of basal-like, proliferative cells. This reflects the ability of papillomaviruses to delay keratinocyte differentiation, thereby maintaining aspects of the basal cell identity of persistently infected cells. This may enable papillomaviruses to establish and maintain long-term infections in squamous epithelial tissues. Previous work has revealed that the ability of ß-HPV8 E6 protein to inhibit Notch and transforming growth factor ß signaling importantly contributes to this activity. Here, we present evidence that HPV8 E6 also subverts Hippo and Wnt signaling and that these activities also aid in restraining keratinocyte differentiation.

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.

Mechanistic Contributions of lncRNAs to Cellular Signaling Pathways Crucial to the Lifecycle of Human Papillomaviruses.

Papillomaviruses are ubiquitous epitheliotropic viruses with double-stranded circular DNA genomes of approximately 8000 base pairs. The viral life cycle is somewhat unusual in that these viruses can establish persistent infections in the mitotically active basal epithelial cells that they initially infect. High-level viral genome replication ("genome amplification"), the expression of capsid proteins, and the formation of infectious progeny are restricted to terminally differentiated cells where genomes are synthesized at replication factories at sites of double-strand DNA breaks. To establish persistent infections, papillomaviruses need to retain the basal cell identity of the initially infected cells and restrain and delay their epithelial differentiation program. To enable high-level viral genome replication, papillomaviruses also need to hold the inherently growth-arrested terminally differentiated cells in a replication-competent state. To provide ample sites for viral genome synthesis, they target the DNA damage and repair machinery. Studies focusing on delineating cellular factors that are targeted by papillomaviruses may aid the development of antivirals. Whilst most of the current research efforts focus on protein targets, the majority of the human transcriptome consists of noncoding RNAs. This review focuses on one specific class of noncoding RNAs, long noncoding RNAs (lncRNAs), and summarizes work on lncRNAs that may regulate the cellular processes that are subverted by papillomavirus to enable persistent infections and progeny synthesis.

Molecular Mechanisms of MmuPV1 E6 and E7 and Implications for Human Disease.

Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes.

Role and Clinical Utility of Cancer/Testis Antigens in Head and Neck Squamous Cell Carcinoma.

Cancer/testis (CT) antigens exhibit selective expression predominantly in immunoprivileged tissues in non-pathological contexts but are aberrantly expressed in diverse cancers. Due to their expression pattern, they have historically been attractive targets for immunotherapies. A growing number of studies implicate CT antigens in almost all hallmarks of cancer, suggesting that they may act as cancer drivers. CT antigens are expressed in head and neck squamous cell carcinomas. However, their role in the pathogenesis of these cancers remains poorly studied. Given that CT antigens hold intriguing potential as therapeutic targets and as biomarkers for prognosis and that they can provide novel insights into oncogenic mechanisms, their further study in the context of head and squamous cell carcinoma is warranted.

The dynamic interactome of microRNAs and the human papillomavirus in head and neck cancers.

The Human Papillomavirus type 16 is a major etiologic factor for a subset of Head and Neck cancers. These cancers of the oropharyngeal region are growing, and it is expected to exceed cervical cancers in the near future. The major oncogenes E6 and E7 mediate many of the early transformation stages targeting p53 and other tumour suppressor genes. The majority of this regulation is centred on protein coding genes but more recently small non-coding RNAs, such as miRNAs are also regulated by HPV16. However, the system-wide impact of HPV16 on miRNAs is yet to be fully understood. To fully gauge the overall relationship between HPV16 and miRNAs, several studies have devised dynamic interactomes which encompass viral oncogenes, miRNAs and gene targets. These interactomes map potential pathways which permit the identification of possible mechanistic links. Our review will discuss the latest developments in using viral interactomes to understand viral mechanisms and how these approaches may aid in the elucidation of potential druggable pathways.

The Mus musculus Papillomavirus Type 1 E7 Protein Binds to the Retinoblastoma Tumor Suppressor: Implications for Viral Pathogenesis.

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts with RB1. We show that MmuPV1 E7 interacts through its C terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting noncanonical activities of RB1, which are independent of RB1's ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis. IMPORTANCE Papillomavirus infections cause a variety of epithelial hyperplastic lesions, or warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas, and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species specific and do not infect heterologous hosts. Here, we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding-defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.

Label-free, High-Resolution Optical Metabolic Imaging of Human Cervical Precancers Reveals Potential for Intraepithelial Neoplasia Diagnosis.

While metabolic changes are considered a cancer hallmark, their assessment has not been incorporated in the detection of early or precancers, when treatment is most effective. Here, we demonstrate that metabolic changes are detected in freshly excised human cervical precancerous tissues using label-free, non-destructive imaging of the entire epithelium. The images rely on two-photon excited fluorescence from two metabolic co-enzymes, NAD(P)H and FAD, and have micron-level resolution, enabling sensitive assessments of the redox ratio and mitochondrial fragmentation, which yield metrics of metabolic function and heterogeneity. Simultaneous characterization of morphological features, such as the depth-dependent variation of the nuclear:cytoplasmic ratio, is demonstrated. Multi-parametric analysis combining several metabolic metrics with morphological ones enhances significantly the diagnostic accuracy of identifying high-grade squamous intraepithelial lesions. Our results motivate the translation of such functional metabolic imaging to in vivo studies, which may enable improved identification of cervical lesions, and other precancers, at the bedside.

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.

Expression of the Long Noncoding RNA DINO in Human Papillomavirus-Positive Cervical Cancer Cells Reactivates the Dormant TP53 Tumor Suppressor through ATM/CHK2 Signaling.

Tumor cells overcome the cytostatic and cytotoxic restraints of TP53 tumor suppressor signaling through a variety of mechanisms. High-risk human papillomavirus (HPV)-positive tumor cells retain wild-type TP53 because the HPV E6/UBE3A ubiquitin ligase complex targets TP53 for proteasomal degradation. While restoration of TP53 in tumor cells holds great promise for cancer therapy, attempts to functionally restore the dormant TP53 tumor suppressor in HPV-positive cancer cells by inhibiting the HPV E6/UBE3A ubiquitin ligase complex have not yet been successful. The damage-induced long noncoding RNA, DINO (DINOL), is a TP53 transcriptional target that has been reported to bind to and stabilize TP53, thereby amplifying TP53 signaling. We show that HPV-positive cervical carcinoma cells contain low levels of DINO because of HPV E6/UBE3A-mediated TP53 degradation. Acute DINO expression overrides HPV16 E6/UBE3A-mediated TP53 degradation, causing TP53 stabilization and increased expression of TP53 transcriptional target genes. This causes a marked sensitization to chemotherapy agents and renders cells vulnerable to metabolic stress. Acute DINO expression in HPV-positive cervical cancer cells induces hallmarks of DNA damage response signaling, and TP53 activation involves ATM/CHK2 signaling. DINO upregulation in response to DNA damage is independent of ATM/CHK2 and can occur in cancer cells that express mutant TP53.IMPORTANCE Functional restoration of the TP53 tumor suppressor holds great promise for anticancer therapy. Current strategies are focused on modulating TP53 regulatory proteins. Long noncoding RNAs (lncRNAs) have emerged as important regulators of TP53 as well as modulators of downstream tumor-suppressive transcriptional responses. Unlike many other cancer types, human papillomavirus (HPV)-positive cancer cells retain wild-type TP53 that is rendered dysfunctional by the viral E6 protein. We show that acute expression of the damage-induced long noncoding RNA, DINO, a known TP53 transcriptional target and functional modulator, causes TP53 reactivation in HPV-positive cervical cancer cells. This causes increased vulnerability to standard chemotherapeutics as well as biguanide compounds that cause metabolic stress. Hence, strategies that target DINO may be useful for restoring TP53 tumor suppressor activity in HPV-positive cancers and other tumor types that retain wild-type TP53.

The Role of Long Noncoding RNAs in Human Papillomavirus-associated Pathogenesis.

Infections with high-risk human papillomaviruses cause ~5% of all human cancers. E6 and E7 are the only viral genes that are consistently expressed in cancers, and they are necessary for tumor initiation, progression, and maintenance. E6 and E7 encode small proteins that lack intrinsic enzymatic activities and they function by binding to cellular regulatory molecules, thereby subverting normal cellular homeostasis. Much effort has focused on identifying protein targets of the E6 and E7 proteins, but it has been estimated that ~98% of the human transcriptome does not encode proteins. There is a growing interest in studying noncoding RNAs as biochemical targets and biological mediators of human papillomavirus (HPV) E6/E7 oncogenic activities. This review focuses on HPV E6/E7 targeting cellular long noncoding RNAs, a class of biologically versatile molecules that regulate almost every known biological process and how this may contribute to viral oncogenesis.

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.

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.

More than just oncogenes: mechanisms of tumorigenesis by human viruses.

Most humans are infected with at least one of the known human cancer viruses during their lifetimes. While the initial infection with these viruses does not cause major disease, infected cells can acquire cancer hallmarks, particularly upon immunosuppression or exposure to co-carcinogenic stimuli. Even though cancer formation represents a rare outcome of a viral infection, approximately one out of eight human cancers has a viral etiology. Viral cancers present unique opportunities for prophylaxis, diagnosis, and therapy, as demonstrated by the success of HBV and HPV vaccines and HCV antivirals in decreasing the incidence of tumors that are caused by these viruses. Here we review common characteristics and mechanisms of action of the human oncogenic viruses.