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Received: October 22^nd 2014

Accepted: May 15^th 2015

Human papillomavirus E7 oncoprotein and its role in the cell transformation

Verónica Vallejo-Ruiz,^a Noé Velázquez-Márquez,^b Patricia Sánchez-Alonso,^c Gerardo Santos-López,^a Julio Reyes-Leyva^a

^aLaboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Metepec

^bFacultad de Medicina, Benemérita Universidad Autónoma de Puebla, Puebla

^cCentro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla

Puebla, México

Communication with: Verónica Vallejo-Ruiz

Teléfono y fax: 24 44 44 01 22

Email: veronica_vallejo@yahoo.com; veronica.vallejor@imss.gob.mx

Human papillomavirus (HPV) genome codifies proteins with oncogenic activity, such as E7. Due to its structural characteristics, the E7 protein may interact with a great variety of cellular proteins. Some of these proteins act as cell-cycle regulators and other proteins function as transcription factors. These interactions play an important role in the induction of mitogenic pathways, in G1/S progression, and the inhibition of cellular differentiation, which increases chromosomal instability. The aim of this study is to describe the interactions of HPV E7 protein with different cellular proteins, and their contribution in the development of cervical cancer.

Keywords: Papillomavirus E7 proteins, Papillomavirus, Cervical cancer.

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The human papillomavirus (HPV) is considered the most common sexually transmitted agent worldwide. Multiple studies have linked infection with HPV the development of cervical cancer (CC) and have considered it the main etiological factor of this cancer.^1,2 HPV prevalence in the general population varies significantly in different parts of the world, from 1.4% to 25.6%.³ These values ​​are increased in women with neoplastic disorders and cancer.^1,4 In fact, over 90% of cases of cervical cancer are related to the presence of HPV.^1,2

The human papillomaviruses have been grouped according to their association with benign or malignant tumors, in low- and high-risk viruses, respectively. The high-risk HPV types include: 16, 18, 31, 33, 35, 39, 45, 52, 58, 59, 67, 68, and 70, of which types 16 and 18 are the most prevalent in malignant lesions of the cervix. On the other hand, types 6, 11, 13, 44, and 74 are considered low-risk because they are generally associated with benign lesions.^2,4

HPV belongs to the Papillomaviridae family, which includes relatively small viruses, non-enveloped, with a diameter of 55 nm. They have an icosahedral capsid consisting of 360 copies of the L1 protein and 12 copies of the L2 protein, organized in 72 capsomeres.⁵

The HPV genome is a circular molecule of double-stranded DNA divided into three regions: a noncoding regulatory region, covering about 10% of the genome and called the long control region (LCR); the region of early expression genes (E), so called because it encodes nonstructural proteins E1, E2, E4, E5, E6, and E7, which are expressed early in infection and are involved in the regulation of viral replication and oncogenesis; and finally, the region containing the late expression genes (L) encoding the L1 and L2 structural proteins, which form the viral capsid.⁶

The oncogenic potential of high-risk HPV resides with the E6 and E7 oncoproteins, which are responsible for disrupting the cell cycle control and initiating the series of changes associated with cellular transformation.^7,8

The expression of viral oncogenes can be regulated by cellular proteins and virus-encoded proteins, such as viral protein E2, which acts as a transcriptional factor. Protein E2 forms homodimers that specifically recognize the ACCGNNNNCGGT palindromic sequences located in the LCR of the viral genome; binding to this site induces transcription of E6 and E7.^9,10 Some cellular proteins may promote the transcription of viral genes, such as the H-ras protein, which increases transcription of oncogenes E6 and E7.¹¹

The viral oncoproteins interact with several proteins that regulate gene expression and affect cell cycle control. Given the importance of the E7 protein of HPV type 16 (the most common in malignant cervical tumors), this review will describe its major structural and functional characteristics and its role in oncogenesis.

The E7 gene of HPV type 16 encodes an acidic nuclear phosphoprotein of 98 amino acids. The E7 protein has structural and functional similarity to the E1A transforming proteins of adenovirus and the T antigen of the poliomaviruses.^12,13 E7 protein has three conserved regions (RC1, RC2, and RC3), so named for the similarity of its amino acid sequence to the E1A protein of the adenovirus. RC1 of E7 consists of residues 1-15 of the amino terminus. RC2 is formed of residues 16-38 and contains the LXCXE motif, which establishes high-affinity interactions with the retinoblastoma protein (pRb).¹⁴ Finally, RC3 consists of residues 39-98 of the carboxyl terminus; this region contains two CXXC motifs, which form a zinc finger structure, allowing it to act as a transcription factor.^15,16 

E7 protein is structurally dynamic, that is, it undergoes conformational transitions that allow it to establish an ordered series of associations with different proteins involved in cell cycle control. The N-terminus is what confers conformational plasticity to the E7 oncoprotein and establishes contact with other proteins.^17,18 These properties of transition and plasticity of E7 play a key role in its ability to interfere with different cellular processes and thereby initiate malignant transformation.

One of the processes that gives rise to cell transformation is the inhibition of pRb by E7 protein of high-risk HPV.¹⁹ pRb protein maintains low cell cycle control, exerting suppressive action on E2F transcription factor, which when it is free promotes the expression of numerous genes involved in progression from phase G1 to phase S of the cell cycle. pRb protein binds to E2F, forming a complex (pRb/E2F) which keeps this transcription factor sequestered during phase G1 of the cell cycle (Figure 1). E7 interaction with pRb leads to dissociation of the pRb/E2F complex, the subsequent degradation of pRb, and premature activation of E2F, which triggers the transcription of numerous genes required by the cell to enter phase S. Among the genes activated by E2F are those encoding cyclins A and E and cyclin-dependent kinases (CDK2). Cyclins are proteins that regulate the transition between different phases of the cell cycle because of their function as kinase triggers.²⁰

It has been reported that E7 protein is capable of inducing the marking of proteins with ubiquitin when interacting with cullin 2, a protein that forms part of the ubiquitin ligase complex. E7 binding with cullin 2 promotes an atypical ubiquitination of proteins of the retinoblastoma family (pRb, p107, and p130), which leads to degradation in the proteasome.^21-24

Not only the E7 protein of high-risk HPV binds to members of the retinoblastoma family; also the E7 protein of low-risk HPV is capable of associating with pRb and p130, which causes a decrease of involucrin, which is a marker of cell differentiation, although the affinity of E7 with pRb is 10 times higher in high-risk HPV than in low-risk.²⁵

Changes in the differentiation process of HPV-infected cells are a result of E7 binding with retinoblastoma proteins, but also of the activity of kinase CKII,²⁶ which plays an important role in the control of mitosis and cellular proliferation.²⁷

Another key target of the E7 oncoprotein of high-risk HPV is the Cdc25A protein. This is a tyrosine phosphatase that is required for the transition from phase G1 to phase S of the cell cycle. Cdc25A removes the phosphate groups that keep cyclin-dependent kinase (CDK2) inhibited, allowing it to then form activation complexes with cyclins A and E.^28,29 E7 protein of HPV type 16 can act on the promoter gene that encodes Cdc25A to induce transcription and increase levels of the protein,30 or it may be associated and directly activate the Cdc25A protein or cyclin A/CDK2 and cyclin E/CDK2 complexes, through different binding sites than it uses to bond to pRb.^30,31   

E7 may also inhibit other factors involved in cell cycle regulation, including the proteins p21cip1 and p27kip1, which repress the action of CDK2.^32,33 All this accords with atypical CDK2 activation in cells expressing E7 protein of HPV type 16; this activation leads to aberrant induction of supernumerary centrosomes and aneuploidy. Induction of abnormalities into mitotic centrosomes is performed in part by association of E7 with gamma-tubulin, the centrosome regulator, and by concomitant alteration of gamma-tubulin assembly in centrosomes, leading to chromosomal instability.³⁴

Another transcriptional repressor inactivated by E7 is factor E2F6, a member of the E2F factor family regulating phase S.³⁵ This factor acts as an independent repressor of retinoblastoma proteins.^20,36 Abundance of E2F6 seems to be lower in cells infected with HPV or expressing E7.³⁵

As mentioned above, E7 protein of HPV dissociates the pRb/E2F complexes that keep transcription factor E2F repressed during phase G1 but, once disassociated, E2F plays a critical role in the transcription of genes that promote cell cycle progression.  

E7 protein also interacts with other transcriptional regulatory proteins such as TATA box binding protein (TBP) and transcription factors AP-1 and c-Myc. E7 interaction with these transcription factors contribute to the process of malignant transformation.

Transcription factor AP1, together with viral protein E2, represents a key factor, though not the only, for inducing transcription of E6 and E7 viral genes. The LCR of high-risk HPV has two AP1 binding sites, one located in the enhancer, and another in the promoter region, as well as binding sites for other transcription factors. Inhibition of the interaction of AP1 with LCR reduces expression of E6 and E7 proteins, and malignant transformation of HPV-infected cells.^37-39 

E7 protein can interact with the AP-1 transcription factor through its "zinc finger" domain, modulating the expression of N-cadherin, among other molecules, via MEK-ERK.⁴⁰

It has also been found that E7 expression causes an increase in c-Myc levels. c-Myc protein is a key transcriptional factor in cell cycle progression, proliferation, metabolism, transformation, and apoptosis.^41-43 c-Myc protein is able to interact and form specific complexes with E7 of HPV type 18, promoting binding of this protein to DNA and increasing the activity of c-Myc transactivation. It has recently been reported that Myc binding ability to telomerase reverse transcriptase promoter increases as the concentration of E7 increases, and E7 protein of HPV 16 cooperates with c-Myc protein to immortalize human keratinocytes.^44,45 The results found show the importance of the effect of E7/c-Myc interaction on various cellular pathways that are of great importance in tumor development.

As previously described, the E7 oncoprotein is necessary for the induction and maintenance of malignant transformation; the development of therapeutic vaccines against cervical cancer has been a challenge in cancer research. One effort has focused on the development of vaccines able to induce a specific immune response against tumor cells. The E7 oncoprotein has represented the ideal target for the development of immunotherapies mediated by cytotoxic T-lymphocyte. Since the E7 protein is a poor inducer of T cytotoxic response, different strategies are being developed to develop vaccines that generate better responses, such as the use of chimeric proteins with E7 protein epitopes coupled to nanoparticles; E7 fusion with a ricin toxin subunit, or development of virus-like particles (VLP) have also represented safe vaccination strategies and both have been carried out.^46-48

Recently, the interaction of various cellular proteins with E7 from 17 different types of HPV was identified through proteomic analysis. This study demonstrated interactions conserved between UBR4/p600 and E7 of different types of HPV, while ENC1 specifically binds E7 of HPV 18 and HPV 45, both from the alpha genus of the species.⁴⁹

Conclusions

The E7 oncoprotein of high-risk HPV has been extensively studied because of its implications for the development of cervical cancer. The importance of E7 is confirmed by the fact that its expression is constant in most HPV-positive malignancies. The structural flexibility of E7 makes it able to bind to different proteins that act as transcriptional regulators, activating or repressing genes whose biological effect will be reflected in cellular processes that contribute to the development of malignant transformation. This important role of E7 in the pathogenesis of HPV infection and oncogenesis makes it the target of therapeutic strategies to fight cancer. At present different ways are being studied to inhibit the function of E7 protein, and it is likely that in the medium term some therapeutic agents will be found that inhibit its effects in cervical cancer patients. 

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