Human papillomavirus-16 and -18 replication in esophagus squamous cancer cell lines does not require sheterologous E1 and E2 proteins

Human papillomaviruses (HPVs) are doublestranded DNA viruses that replicate in the nuclei of squamous epithelial cells. HPVs can be classified into high-risk (e.g., types 16, 18, 31, and 33) or low-risk (e.g., types 6, 11, and 30), depending on their association with benign or malignant tumors. We recently described the association of HPV-16 and -18 with esophagus squamous cell cancer. HPV replication was studied in representative cell lines derived from esophagus cancers. HPV-16 and -18 genomes were independently transiently transfected into HCE-4 and HCE-7 cell lines with and without E1 and E2 genes under heterologous promoters. Southern blot analysis demonstrated that these cell lines support viral replication. However, heterologous E1 and E2 are not required for HPV replication. These findings suggest that specific host nuclear factors in esophageal squamous epithelial cells may support HPV replication. © 1995 Wiley-Liss, Inc.     

Modulation of apoptosis by human papillomavirus (HPV) oncoproteins

Summary. The regulation of host-mediated apoptosis by the E6 and E7 oncoproteins has garnered attention because it is believed to be an important strategy employed by high-risk (HR)-human papillomaviruses (HPVs) to evade immune surveillance. Additionally, the revelation that E5 can protect cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis suggests that it may also play a role in undermining host defense mechanisms. Cellular transformation is an unintended consequence of persistent infection by HR-HPVs, and it is therefore likely that the primary function of E5, E6 and E7 is to regulate cell survival throughout the normal viral life cycle in order to ensure viral replication and promote the spread of progeny. The purpose of this article is to review the literature on the regulation of host-mediated apoptosis by E5, E6 and E7 that describes the mechanisms employed by HR-HPVs to persist in the host and create the conditions necessary for cellular transformation.     

Biology and pathological associations of the human papillomaviruses: a review

Historical cottontail rabbit papillomavirus studies raised early indications of a mammalian DNA oncogenic virus. Today, molecular cloning recognises numerous animal and human papillomaviruses (HPVs) and the development of in vitro transformation assays has escalated oncological research in HPVs. Currently, their detection and typing in tissues is usually by Southern blotting, in-situ hybridization and polymerase chain reaction methods. The complete papillomavirus virion constitutes a protein coat (capsid) surrounding a circular, double-stranded DNA organised into coding and non-coding regions. 8 early (E1-E8) open reading frames (ORFs) and 2 late (L1, L2) ORFs have been identified in the coding region of all papillomaviruses. The early ORFs encode proteins which interact with the host genome to produce new viral DNA while late ORFs are activated only after viral DNA replication and encode for viral capsid proteins. All papillomaviruses are obligatory intranuclear organisms with specific tropism for keratinocytes. Three possible courses of events can follow papillomaviruses entry into cells: (1) viral DNA are maintained as intranuclear, extrachromosomal, circular DNA episomes, which replicates synchronously with the host cell, establishing a latent infection; (2) conversion from latent into productive infection with assembly of complete infective virions; (3) integration of viral DNA into host cellular genome, a phenomenon seen in HPV infections associated with malignant transformation. Human papillomaviruses (HPVs) essentially induce skin and mucosal epithelial lesions. Various skin warts are well known to be HPV-associated (HPVs 1, 2, 3, 7 and 10). Besides HPVs 3 and 10, HPVs 5, 8, 17 and 20 have been recovered from Epidermodysplasia verruciformis lesions. Anogenital condyloma acuminatum, strongly linked with HPVs 6 and 11 are probably sexually transmitted. The same HPVs, demonstrable in recurrent juvenile laryngeal papillomas, are probably transmitted by passage through an infected birth canal. HPVs described in uterine cervical lesions are generally categorized into those associated with high (16, 18), intermediate (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and low (6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 62, 66) risk of cervical squamous carcinoma. Cervical adenocarcinoma, clear cell carcinoma and small cell neuroendocrine carcinoma have also been linked to HPVs, especially HPV18. Other lesions reported to be HPV-associated are: papillomas, dysplasia and carcinomas in the nasal cavity (HPV 6, 11, 57); squamous papilloma, condyloma acuminatum, and verruca vulgaris of the oral cavity (HPV 6, 11), oral focal epithelial hyperplasia (HPV 13, 32); warty lip lesions (HPV 2): and conjunctival papillomas (HPV 6, 11).     

Cellular proteins involved in papillomavirus-induced transformation

Summary Human papillomaviruses (HPVs) are associated with at least 80% of cervical carcinomas and are classified as high-risk or low-risk based on whether or not they are commonly found in cervical cancers. The high-risk HPVs have early gene products (E6 and E7) that immortalize human keratinocytes and are at least partially responsible for causing cervical carcinoma. E6 and E7 from the high-risk viruses interact strongly with the tumor suppressors p53 and Rb; those from the low-risk HPVs do not. Transformation involves a multi-step process and requires additional factors besides high-risk HPV infection. High-risk HPVs are capable of immortalizing primary human keratinocytes in tissue culture, but such cells become transformed only after certain chromosomal changes take place, possibly having to do with oncogene activation. The DNA of high-risk HPVs is frequently (if not always) integrated into the genome of cancer cells; it is normally episomal in premalignant lesions. Integration disrupts the E2 and E5 genes and viral gene regulation. Cells containing integrated viral DNA show excessively high levels of E6 and E7. While there is some conflicting evidence, it appears that the p53 and Rb tumor-suppressor genes are more frequently mutated in HPV-negative tumors than they are in HPV-positive tumors, suggesting that for tumor formation to proceed the p53 and Rb proteins must be inactivated either by interaction with the viral proteins or by mutation. The presence of an activated oncogene in a cell lacking functional p53 or Rb may then be sufficient to cause tumor progression.     

Association of human papillomavirus with Fanconi anemia promotes carcinogenesis in Fanconi anemia patients

Fanconi anemia (FA) is a rare recessive disorder associated with chromosomal fragility. FA patients are at very high risk of cancers, especially head and neck squamous cell carcinomas and squamous cell carcinomas caused by infection of human papillomaviruses (HPVs). By integrating into the host genome, HPV oncogenes E6 and E7 drive the genomic instability to promote DNA damage and gene mutations necessary for carcinogenesis in FA patients. Furthermore, E6 and E7 oncoproteins not only inhibit p53 and retinoblastoma but also impair the FANC/BRCA signaling pathway to prevent DNA damage repair and alter multiple signals including cell‐cycle checkpoints, telomere function, cell proliferation, and interference of the host immune system leading to cancer development in FA patients. In this review, we summarize recent advances in unraveling the molecular mechanisms of FA susceptibility to HPV‐induced cancers, which facilitate rational preventive and therapeutic strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

Cellular transformation by human papillomaviruses: lessons learned by comparing high- and low-risk viruses

The oncogenic potential of papillomaviruses (PVs) has been appreciated since the 1930s yet the mechanisms of virally-mediated cellular transformation are still being revealed. Reasons for this include: a) the oncoproteins are multifunctional, b) there is an ever-growing list of cellular interacting proteins, c) more than one cellular protein may bind to a given region of the oncoprotein, and d) there is only limited information on the proteins encoded by the corresponding non-oncogenic PVs. The perspective of this review will be to contrast the activities of the viral E6 and E7 proteins encoded by the oncogenic human PVs (termed high-risk HPVs) to those encoded by their non-oncogenic counterparts (termed low-risk HPVs) in an attempt to sort out viral life cycle-related functions from oncogenic functions. The review will emphasize lessons learned from the cell culture studies of the HPVs causing mucosal/genital tract cancers.     

The biological properties of E6 and E7 oncoproteins from human papillomaviruses

More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV types.     

Biology of human papillomaviruses

Human papillomaviruses (HPVs) cause squamous cancers of epithelial surfaces, of which genital cancers are the most common. In this article we have attempted to describe the properties and functions of the viral proteins of HPV type 16, a common cause of genital cancers, and have tried to suggest how their expression may lead to a dysregulated cell which may become malignant. These viruses are attempting to replicate in terminally differentiating keratinocytes and must stimulate G1 to S-phase progression for the replication of their genome. As part of the successful completion of replication and assembly of infectious virus particles, the virus needs at least partial differentiation to occur. Therefore, at the same time as differentiation is occurring, the nuclei of infected cells are in S-phase. While the mechanisms of action of the viral proteins are not completely understood, researchers are making progress and this article strives to bring together the conclusions from some of this work.     

Lytic infection by double-strand DNA viruses and cell cycle alterations

Polyomaviruses, papillomaviruses, adenoviruses and herpesviruses are double-stranded DNA viruses that replicate in the nucleus of the cells they infect and have evolved various strategies to create a cellular environment that is optimally conducive to their replication. One of these strategies consists of activating cellular genes, mostly S-phase genes that are required for the replication of the viral genome. Concomitantly, they encode one or several proteins that negatively regulate the response of the cell to viral infection, notably cell cycle arrest and/or apoptosis. As a result, these viruses profoundly alter the biochemical pathways that normally control cellular growth, and may thus promote uncontrolled cell proliferation. This review describes some well-known mechanisms of cell cycle alteration induced by these viruses.     

The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2

The high risk human papillomaviruses (HPVs) are associated etiologically with the majority of human cervical carcinomas. These HPVs encode two viral oncoproteins, E6 and E7, which are expressed consistently in cervical cancers. The function of these viral oncoproteins during a productive infection is to ensure viral replication in cells that have normally withdrawn from the cell division cycle and are committed to terminal differentiation. Expression of the E7 oncoprotein has been shown to lead to the abrogation of various negative growth regulatory signals, including a p53-mediated G₁ growth arrest, TGFβ-mediated growth inhibition, and quiescence of suprabasal keratinocytes. Here we describe a novel mechanism by which E7 can uncouple cellular proliferation and differentiation. In contrast to normal, differentiating keratinocytes, HPV-16 E7-expressing keratinocytes show delayed cellular differentiation and elevated cdk2 kinase activity despite high levels of p21^Cip1 and association of p21^Cip1 with cdk2. We show that the HPV E7 protein can interact with p21^Cip1 and abrogate p21^Cip1-mediated inhibition of cyclin A and E-associated kinase activities. Based on these findings, we propose that this capacity of the HPV E7 oncoprotein to overcome p21^Cip1-mediated inhibition of cdk2 activity during keratinocyte differentiation contributes to the ability of E7 to allow for cellular DNA synthesis in differentiated keratinocytes.     

Papillomavirus E1 Proteins: Form,Function, and Features

The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.     

Aetiology, pathogenesis, and pathology of cervical neoplasia.

Early epidemiological studies of cervical neoplasia suggested a causal relation with sexual activity and human papillomaviruses (HPVs) have emerged as prime suspects as venerally transmitted carcinogens. HPVs fall into two broad camps: low risk types, associated with cervical condylomas and CIN 1; and high risk types (mostly 16 and 18), found in 50-80% of CIN 2 and CIN 3 lesions, and 90% of cancers. This association with cancer is very strong, with odds ratios of > 15 (often much higher) in case-control studies that are methodologically sound. An infrequently detected third group of intermediate risk type HPVs is associated with all grades of CIN and occasionally with cancers. HPVs have also been detected in a wide range of asymptomatic controls, indicating that other events are required for development of neoplasia such as viral persistence and/or altered expression of viral genes, often following integration of the viral genome. This leaves the two major viral oncogenes, E6 and E7, directly coupled to viral enhancers and promoters, allowing their continued expression after integration. High risk HPV E7 proteins bind and inactivate the Rb protein, whereas E6 proteins bind p53 and direct its rapid degradation. A range of putative cofactors has been implicated in progression: HLA type, immunosuppression, sex steroid hormones, and smoking; most of these cofactors appear to influence progression to CIN 3. The natural history includes progression to CIN 3 in 10% of CIN 1 and 20% of CIN 2 cases, whereas at least 12% of CIN 3 cases progress to invasive carcinoma. Cervical glandular intraepithelial neoplasia (CGIN) often coexists with squamous CIN, and the premalignant potential of high grade CGIN is not in doubt, but the natural history of low grade CGIN remains uncertain. A high proportion of CGIN lesions and adenocarcinomas are HPV positive, and HPV18 has been implicated more in glandular than in squamous lesions. A strong clinical case for the application of HPV typing of cells recovered from cervical scrapes can be made; however, a rigorous cost-benefit analysis of introducing HPV typing into the cervical screening programme is required. Prophylactic and therapeutic HPV vaccines are under development. This article reviews the aetiology, pathogenesis, and pathology of cervical neoplasia, emphasising the role of HPVs.     

Human papillomavirus proteins as prospective therapeutic targets

Human papillomaviruses (HPV) are the causative agents of a subset of cervical cancers that are associated with persistent viral infection. The HPV genome is an ∼8 kb circle of double-stranded DNA that encodes eight viral proteins, among which the products of the E6 and E7 open reading frames are recognized as being the primary HPV oncogenes. E6 and E7 are expressed in pre-malignant lesions as well as in cervical cancers; hence these proteins have been extensively studied as potential targets for HPV therapies and novel vaccines. Here we review the expression and functions of E6 and E7 in the viral vegetative cycle and in oncogenesis. We also explore the expression and functions of other HPV proteins, including those with oncogenic properties, and discuss the potential of these molecules as alternative therapeutic targets.     

An overview of human papillomaviruses and current vaccine strategies

Cervical cancer is one of the most common cancers in women worldwide, particularly in developing countries. The viral origin of cervical cancer has been proven beyond any reasonable doubt. Persistent infection with certain subsets of human papillomaviruses is recognized as a necessary cause for the development of cervical cancer. Persistence of oncogenic HPVs, immunodeficiency, high HPV viral load and cofactors like smoking, multiple sex partners and poor nutrition predispose to cervical cancer. Prophylactic vaccines using HPV virus-like particles containing capsid protein L1 have shown protection against disease in animals and are currently undergoing clinical trials. Therapeutic vaccines using HPV E6 and E7 proteins are also being investigated for their ability to remove residual infection.