- Open Access
Papillomavirus E5: the smallest oncoprotein with many functions
Molecular Cancer volume 10, Article number: 140 (2011)
Papillomaviruses (PVs) are established agents of human and animal cancers. They infect cutaneous and mucous epithelia. High Risk (HR) Human PVs (HPVs) are consistently associated with cancer of the uterine cervix, but are also involved in the etiopathogenesis of other cancer types. The early oncoproteins of PVs: E5, E6 and E7 are known to contribute to tumour progression. While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems. The Bovine PV (BPV) system has been the most useful animal model in understanding the oncogenic potential of PVs due to the pivotal role of its E5 oncoprotein in cell transformation. This review will highlight the differences between HPV-16 E5 (16E5) and E5 from other PVs, primarily from BPV. It will discuss the targeting of E5 as a possible therapeutic agent.
PVs are established agents of human and animal cancers . They infect cutaneous and mucous epithelia inducing benign tumours which usually regress. Occasionally, the tumours progress to malignancy. Over 120 types of HPVs have been identified so far and among these 15 have been defined as HR HPVs. These are consistently associated with cancer. Genital HPVs are sexually transmitted and HR genital HPVs are a necessary factor in the development of almost all cases of cervical cancer. HPV-16 and -18 are the viruses most frequently associated with cancer of the uterine cervix (CxCa) .
CxCa is the second most common cancer in women worldwide killing about 0.25 million women per year. However, in economically developed countries the rate of CxCa is dramatically reduced due to screening program based on exfoliative cervical cytology (PAP smears).Vaccines to prevent HR HPV infection are available, although their use should be implemented along with screening programmes to further reduce the incidence of such cancer . HR HPVs are also involved in the etiopathogenesis of other anogenital cancer . Furthermore HPV, particularly HR HPV-16 is strongly associated to oral squamous cell carcinoma and other potentially malignant oral lesions . Growing evidence also suggests that HR HPV-16 is involved in the etiopathogenesis of head and neck squamous cell carcinomas, suggesting that HPV vaccines should be also considered for prevention of this type of cancer [5, 6]. Additionally, HPVs may be involved in the etiopathogenesis of others cancer types, including tumours of the upper respiratory tract, eye, esophagus, non-small-cell lung cancers [7–10]. The presence of HPV-16 has been reported also in colorectal carcinoma , breast cancer  and urinary bladder carcinoma . Recently, HPV DNA has been associated also with prostatic tumours . The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems which often provide new and profitable avenues of research . The BPV system has been one of the most useful animal models in understanding the oncogenic potential of PVs. Furthermore, the mechanisms by which BPV induces tumors are an outstanding model to better understand the pathogenesis of other cancer types. The importance of the role of HPV in cancer etiology and development has been recognized by the assignment of the 2008 Nobel Prize for Medicine to Prof. Harald zur Hausen who firstly observed that infection with HPVs is responsible for CxCa development. The genome of PVs is a double stranded circular DNA roughly divided into three parts: the E region coding for early proteins (E1-E7) responsible for the pathogenicity of the virus; the L region coding for late structural proteins (L1, L2) and a non coding region which contains the cis-elements necessary for replication and transcription of the viral genome. Both in vivo and in vitro studies have pointed to E6 and E7 as the main HPV oncogenes, whereas E5 is the major oncogene of BPV. The E6 oncoprotein interacts with the cellular tumour suppressor p53  and directs its degradation . The primary target of the E7 oncoprotein is the retinoblastoma (Rb) proteins, the inactivation of which leads to tumour progression . Both E6 and E7 also interact with many others cellular factors inducing genomic instability, tumour progression and immune evasion .
While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. However, recent studies have highlighted the important role of this oncoprotein in cell transformation, tumourigenesis and immune modulation, thus implicating E5 in pivotal steps of carcinogenesis. Because of the perceived growing role of E5 in infection establishment and in cell transformation, it is worth considering the most salient features of the oncoprotein. This review will focus on the activities of E5, in particular from HPV-16, and from BPV. Targeting E5 as a possible therapeutic agent will also be briefly discussed.
E5 and the evolution of HPVs
HPVs have been classified into five genera (Alpha-, Beta-, Gamma-, Mu- and Nu-PVs), but not all HPV genera code for an E5 protein . For instance αHPVs encode and express E5 but β HPVs do not. A phylogenetic analysis of known genital HPV types (derived using a Bayesian methodology, with notation of carcinogenic risk levels assigned by the large case-control study conducted by the International Agency for Research on Cancer ), suggested that at least 3 ancestral papillomaviruses are responsible for the current heterogeneous group of genital HPVs. The three major groups that emerged include alpha papillomavirus species and interestingly all carcinogenic types derive from a common ancestor and can code for an E5 protein, whereas the other HPVs either lack a definable E5 ORF (Open Reading Frame) or a translation start codon for E5. As notable exceptions, 10 types which cause benign venereal warts, can also code for an E5 protein. In addition, 16E5 variants with the greatest mitogenic activity in vitro are most frequently detected in the population and most commonly associated with cervical lesions.
The HPV E5 ORF itself has been classified into four different groups: alpha, beta, gamma and delta , which correlate with different clinical manifestations, in particular with oncogenic potential . Thus, the E5-alpha protein is encoded by HR αHPV, whereas the E5-gamma and E5-delta proteins are encoded by low-risk genital HPVs . The phylogenetic analysis suggests that E5 must give some advantage to the virus expressing it and variants of this protein appear to increase the likelihood of oncogenic transformation following persistent infections. However, the E5 ORF is absent in the genome of many HPVs, such as beta-, gamma- and mu-HPVs, indicating that the protein is not essential for the life cycle of these viruses but rather can give some ADDED VALUE to favour infection and transformation (Figure 1). The HPV E5 ORF is expressed during the early phases of the viral life cycle but only as the fourth ORFs on polycistronic transcripts. Since HPVs are thought to use a leaky ribosome-scanning mechanism to translate proteins from polycistronic mRNAs, little E5 protein is likely to be synthesized from these transcripts. In contrast, on epithelial-cell differentiation, E5 is expressed as the second ORFs of late transcripts. It is therefore likely that E5 is synthesized highly in differentiating suprabasal epithelial cells  (Figure 1). In agreement, the BPV-1 E5 protein was detected at a low uniform level in basal layers and at a higher level in the uppermost layers of stratified squamous epithelium in papillomas productively replicating BPV-1 . In addition, E5 was also shown to be expressed in basal and suprabasal layers of BPV4-induced papillomas  and of HPV-16 induced cervix lesions .
In contrast to the highly transforming BPV-1 E5, the HPV E5 proteins display weak transforming activity in vitro (Table 1). Experiments with HPV-6 provided the first evidence that a HPV E5 protein had transforming activity in mammalian cells, as expression of HPV-6 E5 in established murine fibroblasts lead to anchorage independent growth . Later it was shown that also 16E5 induces anchorage independence, more efficient growth in low serum and tumorigenic transformation of murine keratinocytes and fibroblasts [30–32]. In addition, the acute expression of 16E5 stimulates cellular DNA synthesis in primary human keratinocytes, and in cooperation with E7, induces proliferation of primary rodent cells [33–36]. The transforming activity of E5 from HPV-59 and rhesus papillomavirus has been demonstrated in various cell types and assays [37, 38].
Structure and cell interaction
16E5 is 83 amino acid long. The detection of this protein has proved very difficult given its extreme hydrophobicity, membrane localisation and very low levels of expression. For these reasons expression of E5 is often inferred from the presence of E5 mRNA. 16E5 has been detected by immunohistochemistry by Chang et al.  in low-grade squamous intraepithelial lesions (LSILs), in high-grade SILs, CIN (cervical intraepithelial neoplasia) and paradoxically in cancer lesions although often this gene is deleted during viral integration into the host genome. Amino acid sequence analyses of 16E5 suggest it comprises 3 anchor-like α-helices (residues 8-30, 37-52 and 58-76), with only the first being long enough to span a lipid bilayer.
When over-expressed, 16E5 is present in the endoplasmic reticulum (ER), in the nuclear envelope and in the Golgi apparatus (GA) , whereas at a more physiological level of expression, as shown in primary human foreskin keratinocytes, it localizes almost exclusively to the ER and to lesser extent to GA and early endosomes . Recently, it was reported that in HaCaT cells about 10% of total 16E5 can localize to plasma membrane with intracellular amino terminus and extracellular carboxyl-terminus. This observation suggests a fusogenic role of 16E5 with the induction of cell-cell fusion and the formation of binucleated HaCaT cells [41–43]. In contrast, other authors report the presence of 16E5 protein only in the ER of COS and ectocervical cells in the opposite orientation: intraluminal amino-terminus and cytoplasmic carboxyl-terminus . The cytoplasmic localization of the C terminus is further strengthened by the reported interaction of 10 amino acids at the C terminus of 16E5 with karyopherin β3 (KNβ3) , an abundant cellular protein localized mainly in the cytoplasm [46, 47]. An explanation for these contrasting data is the possible presence of 16E5 in the plasma membrane as a consequence of the over-expression of 16E5 by the adenovirus vector system used in HaCaT cells. In agreement, when BPV-1 E5 is expressed at very high levels, such as in baculovirus-infected cells, it is detectable on the plasma membrane in addition to the ER and GA .
16E5 self-associates in vitro and in vivo[40, 50] and this oligomerization takes part mostly by hydrophobic interaction. The reported disulfide bonding  appears less likely because models of 16E5 membrane topology predict the localization of all six cysteine residues within the lipid bilayer [21, 51] rendering cysteine dimerization more difficult.
E5 and cell transformation
E5 interacts with a number of cellular proteins and these interactions are deemed important for the biological activity of the protein in cell transformation and evasion of the immune response. Thus the first transmembrane domain of 16E5 and HPV-31E5 interacts directly with the heavy chain component of the MHC I (Major Histocompatibility Complex class I) via the leucine pairs present in this region; interestingly this same transmembrane domain interacts with a chaperone of MHC I, Bap31 [52, 53]. A region within the second helix (residues 41-54) may be the binding site for the 16 kDa pore sub-unit of vacuolar-ATPase (V-ATPase) , although others claim this is located between residues 54-78 .
E5 is a weak transforming protein in vitro and its effects are seen best when in co-operation with the other viral oncoproteins: 16E5 together with E6 can induce the formation of koilocytes, large cells with cleared cytoplasm and pyknotic nuclei with inconspicuous nucleoli, a well known morphological marker of HPV infection . The development of koilocytotic vacuoles may be linked to the E5-induced relocalization of calpactin I to the perinuclear region promoting perinuclear membrane fusion . Hu et al.  confirmed previous reports describing the oncogenic capacity of 16E5 [30, 58] and offered mechanistic insights into how E5 expression brings about morphological changes in the cervical epithelium. Further, by identifying endoreplication as the mechanism by which the aberrant nuclei form and increased DNA synthesis arises, there is now a biological process that can be targeted to inhibit oncogenesis . Fusogenic 16E5 is expressed on the plasma membrane of cells and both cells must express E5 for cell-cell fusion to occur, indicating that E5 cannot induce cell-cell fusion through an interaction with another protein (receptor) on a neighbouring cell, but rather forms E5-E5 dimers or complexes containing at least two 16E5 molecules. These findings provide important insight into how the fusogenic process is mediated .
E5 of HR HPV by itself can induce morphological and chromosomal changes that frequently accompany the progression of normal cells to cancerous cells. Increased nuclear size, increased DNA content and tetraploidy are characteristic of LSIL [59, 60]. All of these morphological changes have been detected in cell expressing 16E5 and, in particular, the aberrant nuclei formation seems to be due to endoreplication rather than a consequence of cell-cell fusion and failed cytokinesis . Many of these changes are criteria used in the clinical detection of cervical cancer precursors in screening Pap tests . Other morphological changes, such as the presence of binucleated cells, increased DNA content and polyploidy , are not used in the diagnosis of precancerous lesions despite being less subjective because they require larger amounts of material, time, and expenses.
HPV E5 interaction with host cell
HPVs infect stratified epithelia, and their whole life cycle is inextricably linked to keratinocyte differentiation and, in order to establish a persistent infection, HPVs have evolved to overcome many "obstacles":
The stratified epithelium is associated with continuous cellular turnover and desquamation of the terminally differentiated keratinocytes, thus maintenance of the papillomavirus within the tissue requires the infection of basal epithelial cells and the propagation of these infected cells.
Papillomaviruses use the cellular machinery for their replication and need to maintain cell division together with a delayed but not completely inhibited differentiation. HPV infected keratinocytes with imbalanced DNA synthesis/differentiation are forced to apoptosis by the cellular control mechanism.
To establish persistent infection HPV must fight or evade immune-surveillance.
It is clear that the timely and epithelial differentiation-dependent expression of all E proteins is essential to favour viral replication and in turn to overcome the above mentioned obstacles. The "E5 added value" will be highlighted in the following paragraphs.
E5, growth factors and cell cycling
HPV infection is thought to take place when epithelial basal cells are directly exposed to the virus during microinjuries . Infection of epithelial stem cells, which have the capacity for self-renewal, can ensure long term maintenance of the viral genome. During the productive stage of the HPV life cycle, the early viral proteins are expressed, maintaining 50-100 copies of episomal DNA per cell by synchronous replication with host cell DNA [64–66]. The demonstrated ability of 16E5 to enhance ligand-dependent activation of the EGF-R [56, 58–60] (Figure 2) and to stimulate EGF-dependent proliferation of cultured human keratinocytes [33, 35, 58] suggests that 16 E5 may play a major role in expanding populations of HPV-16-infected basal keratinocytes in vivo by enhancing ligand-dependent EGF-R activation. Confirmation of this E5 activity comes from a study on 16E5 transgenic mice showing that functional EGF-R is required for the induction of epidermal hyperplasia and formation of spontaneous skin tumours . 16E5 oncoprotein binds and inhibits the activity of the 16 kDa subunit of V-ATPase, altering the endosomal acidification and degradation of EGF-R [34, 68] thus enhancing its recycling to the plasma membrane. 16E5 can also delay EGF-R degradation by interfering with membrane trafficking and the fusion of early and late endosomes . Recently, 16E5 was shown to form a complex with KNβ3 which is localized mainly in the cytoplasm near the nuclear envelope . It has been suggested that a KNβ3/16E5 complex plays an important role in vesicle trafficking, reinforcing the hypothesis that E5 has a central role in altering protein trafficking inside the cell. The EGF-R signalling pathway can be activated by 16E5 through either EGF-dependent or EGF-independent processes. 16E5 activates mitogen-activated protein kinase (MAPK) p38 and ERK1/2 in human keratinocytes in an EGF-independent manner . Two different pathways, a receptor tyrosin kinase-mediated pathway and a protein kinase C (PKC)-dependent pathway, are involved in the MAPK activation [71, 72] which increases the transcription of c-fos and c-jun[33, 73, 74], forcing the cells through the cell cycle and stimulating transcription of the viral oncogenes E6 and E7. In contrast with EGF-R data, less is know about the role of the interaction of 16E5 with other ErbB family members such as ErbB2 or ErbB4 receptor [28, 75–77].
16E5 is also capable of interacting with, and enhancing the signalling of, different classes of growth factor receptors like the G protein-coupled endothelin receptor (ETA) . In growth factor-starved keratinocytes 16E5 enhances the mitogenic activity of endothelin-1 (ET-1) the specific ligand of ETA (Figure 2). Interestingly it was reported that the mitogenic activity of ET-1 may lead to the chronic stimulation of keratinocyte proliferation observed in psoriasis, an inflammatory/proliferative disorder of the skin, suggesting an important role for ET-1 in epithelial proliferation . Furthermore cross-talking between ETAR and EGF-R pathways would have an amplifying effect on cell proliferation .
In contrast to the activation of EGF-R, 16E5 can down-modulate the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGF-R/FGF-R2b), through reduction of transcripts and protein with alteration of the receptor endocytic trafficking. This causes a decrease in the growth response to the receptor ligands, suggesting that 16E5 might have a role on HPV infection by perturbing the KGFR-mediated physiological behaviour of confluent keratinocytes committed to differentiation . Thus, 16E5 could exert two opposite effects, both related to virus replication: (a) increasing the growth of the most basal undifferentiated cells by up-regulating the EGF-R pathway and (b) decreasing the physiological proliferation/differentiation of the suprabasal keratinocytes by down-modulation of KGFR expression and signalling.
16E5 can also down-regulate the expression of tumour suppressor p21 and p27, both of which are cyclin-dependent protein kinase inhibitors (CKIs), thus causing cell cycle progression and DNA synthesis (S-phase) (Figure 2). The p21Wafl/Sdil/Cipl down-regulation is at transcriptional level  whereas that of p27Kip1, one of the most abundant CKIs, is through reduction of the half-life of p27Kip1 protein .
Other biological activities of 16E5 contribute to the proliferation of the infected cells, such as the interference with cell-cell communication and alteration of adherence and cell motility. The ability of 16E5 to inhibit gap junction-mediated communication between epithelial cells in monolayer  and in raft cultures  by interfering with connexin 43 may render the transformed cells more insensitive to homeostatic growth control signals from adjacent normal cells.
E5 and apoptosis
Due to the integration of HR HPV genome during malignant progression, the E5 gene is not expressed in cervical tumours but both 16/18 E5 mRNA and protein have been detected in anogenital LSIL [85, 86] supporting the possibility that E5 plays a role in early steps of HPV infection to protect infected cells from apoptosis. Indeed, it has been proposed that inhibition of death receptor-mediated apoptosis in human keratinocytes, needed to prevent apoptosis at early stages of viral infection, is a primary function of the HR HPV E5 protein. 16E5 impairs tumor necrosis factor ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT cells by: (a) downregulating the total amount of Fas receptor and reducing Fas surface location; and (b) altering the formation of Death-Inducing Signalling Complex (DISC) triggered by TRAIL . Raft cultures of 16E5-expressing keratinocytes were completely protected from FasL- or TRAIL-induced cell death . Likewise, when UV radiation was used to induce stress, E5-expressing human keratinocytes were protected from apoptosis . In contrast, 16E5 sensitizes human keratinocytes to apoptosis induced by osmotic stress, perhaps due to cell membrane modifications caused by this strongly hydrophobic molecule .
E5 and ER stress pathway
The presence of viral proteins may activate cellular defence mechanisms and in particular the ER stress response. 16E5 can suppress three key proteins of the ER stress pathway: cyclooxygenase-2 (COX-2), XBP-1 and IRE1a. As it was suggested for other viruses, the down-regulation of these proteins can favour viral persistence  which is a major contributory factor to the development of cancer by high-risk HPVs . The inhibition of the ER stress pathway by E5 seems to be limited to the high risk types (Table 1); HPV-6b E5 is unable to alter XBP-1  and increased levels of COX-2 were reported in recurrent respiratory papillomatosis (RRP) lesions caused by low-risk HPV type 6b and 11 [94, 95]. 16E5 is also able to lower COX-2 expression in cells co-expressing E6/E7, suggesting that it might exert similar activity during viral replication.
However, in different cell systems and clinical conditions the same viral early genes seem to exert opposite effects such as the induction of COX-2 expression in spontaneously immortalized HaCaT and cervical cancer lines C33A and SiHa [96, 97]. It is possible that the different cell origin, immortalization and transformation status, and the relative expression of the HPV early proteins account for these contrasting data. Nevertheless the reported consistent down-regulation of ER stress response genes by 16E5 in primary genital keratinocytes leads to speculation that this ER stress pathway inhibition is an event favourable to viral replication and persistence .
Finally, 16E5 may induce expression of one of the Prostaglandin E2 (PGE2) receptor in cervical cancer cells by stimulating the binding of CREB to a variant CRE site in the promoter of EP4 gene  (Figure 2). EP4 pathway activates protein-kinase A which mediates ubiquitin-proteasome-mediated Bax degradation, inducing antiapoptotic effects. Activation of EP4 by 16E5 increases anchorage-independent colony formation and vascular endothelial growth factor (VEGF) expression, leading to speculation that al least in some tumours E5 is involved in tumour growth, angiogenesis and metastasis by inducing inflammatory cell signalling pathways.
HPV E5 and immune evasion
Cervical carcinogenesis is a multi-step process which starts with viral infection; one of the steps is persistence of viral infection. At least three major factors may favour papillomavirus persistence: the virus life cycle takes place away from dermal immune cells , the virus does not cause cell lysis and therefore no or weak inflammatory response , and, finally, the viral proteins actively fight the immune response . The E6 and E7 proteins play an important role in this fight but, once again, the E5 protein seems to add help to the virus by down-regulating MHC/HLA class I. MHC class I is much reduced on the cell surface and accumulates in the GA in cells expressing 16E5 . The arrest of MHC class I in the GA is due to E5-induced alkalinisation of the endomembrane compartments , following 16E5-16 k subunit c interaction, and to the direct interaction of E5 with the heavy chain of the MHC class I complex [53, 104]. These conclusions have been reached in cultured HaCaT cells and W12 cell line, and in vivo in the bovine model . The functional effect of the decreased expression of HLA is a reduction of the recognition by CD8+ T cells in vitro .
Furthermore consistent with its role in the alkalinization of endosomes, 16E5 can prevent the endosomal breakdown of the invariant chain, a chaperone important in the maturation of HLA class II, leading to inhibition of expression of surface HLA class II .
Inhibition of HLA class I transport by 16E5, in contrast to inhibition by BPV E5, is reversible by interferon (IFN) treatment. IFN is sufficient to overcome the inhibitory effect on MHC transport in presence of low levels of 16E5. However, in oncogenic HPV infections, E6 and E7 can inhibit the type I IFN pathway [108–110], thus preventing the IFN-mediated release of E5-induced blockage of HLA class I traffic.
Although an efficient mechanism to avoid cytoxic T lymphocytes (CTL)-mediated immune clearance, the reduction or absence of surface MHC class I renders cells more susceptible to Natural Killer (NK) cell attack. Human NK cells express multiple receptors that interact with HLA class I molecules, including killer cell immunoglobulin-like receptors (KIRs) that predominantly recognize classical HLA class I, including HLA-C, and the C type lectin superfamily of receptors that specifically interact with the nonclassical class I molecule HLA-E. Recognition of the class I molecules by their inhibitory receptors inhibits NK-mediated cell lysis, which would occur in the absence of HLA-C/E. Thus to efficiently evade immunosurveillance, HPV has to selectively down-regulates the HLA class I molecules. Indeed 16E5 selectively inhibits surface expression of HLA-A and HLA-B without affecting either synthesis or transport to the cell surface of HLA-C/E [97, 100, 101]. In this way HPV-16 is potentially capable of avoiding both CTL and NK cell killing.
The bridging of innate and adaptive immune responses can also be affected by E5 via the inhibition of CD1d-mediated cytokine production that would otherwise occurs upon interaction between cell surface CD1d and iNKT cells .
It was reported that interactions between 16E5 and calnexin interfere with modification of HLA class I HCs and results in heavy-chain retention in the ER . Since the synthetic pathways for CD1d and HLA class I HCs are similar, this E5-calnexin interaction may alter CD1d trafficking. Indeed, interactions between 16E5 and calnexin do not appear to interrupt all of the functions of calnexin, but just enough to coopt the cellular cytosolic proteolytic pathway and degrade CD1d. Thus E5 inhibits the CD1d-mediated innate and adaptive immune pathways early in HPV infection.
Finally, the reported novel association of HPV-16 and HPV-31 E5 with Bap31 and A4 can also have an effect on immune escape . Bap31 is a chaperone involved in quality control of, for instance, MHC molecules; A4 is a putative ion channel protein of the endoplasmic reticulum. E5 and Bap31 physically interact and colocalise in perinuclear structures and it was demonstrated that this binding correlates with the ability to retain the proliferative capacity of infected keratinocytes following differentiation. E5 also binds and colocalises with A4 independently of Bap31, however the biological significance of this interaction remains to be established. Interestingly 16E5 first transmembrane domain which binds MHC heavy chain shows homology with the third transmembrane domain of Bap31 . Therefore it is possible that membrane-bound 16E5 displaces Bap31 from MHC I, maybe taking advantage of its own interaction with Bap31, and thus retains MHC I in the ER/GA.
Thus immune escape mediated by E5 seems to be a complex process, perhaps evolved to impact on different pathways regulating the intracellular trafficking of immunosurveillance molecules by general not-specific mechanisms, such as endosomal alkalization, and specific ones, such as binding to MHC I heavy chain and chaperones such as calnexin, invariant chain or Bap31. It remains to be seen if E5 expression causes immune escape also in vivo. An inverse correlation exists between expression of 16E5 and presence of surface MHC I in the W12 keratinocyte line derived from a CIN I biopsy , and in a small panel of naturally occurring CIN lesions (Campo, unpublished observations). Additionally, again in a limited number of samples, an association has been reported between decreased CD1d immunoreactivity and progression of cervical neoplastic lesions with statistical significance (P = 0.0001) .
Cell transformation pathways alternative to E5
Beta-HPVs do not code for an E5 protein. Considering the ubiquitous presence of beta-HPVs in the healthy population with almost no associated pathologies, the lack of E5 ORF in these HPV could represent a factor hampering the fully replicative cycle of these viruses. Moreover, some beta-HPVs are detected together with HPV-3 or related genotypes , and such a co-detection with E5-encoding HPVs suggests that beta-HPVs benefit from E5 delivered by the co-infecting HPV . In human pathology there is a rare, autosomal recessive genodermatosis associated with a high risk of skin carcinoma, Epidermodysplasia Verruciformis (EV), characterized by abnormal susceptibility to infection by beta HPVs. Genetic analysis led to the identification of two adjacent genes (EVER1 and EVER2), the mutation of which segregates with the disease . This extraordinarily high sensitivity to infections by cutaneous beta-HPVs in otherwise-healthy individuals carrying a mutation in one of the EVER genes suggests that in humans a natural EVER-based barrier exists, which protects the host from PVs. The EVER proteins are crucial for the functional integrity of the EVER/ZnT-1 complex [115, 116] responsible for maintaining a low level of free zinc, modulating the activity of the AP-1 transcription factors needed for viral genome expression. It has been proposed that a cellular zinc imbalance constitutes an important, perhaps even a crucial, step in the HPV life cycle. It seems that breaking the barrier to cellular zinc balance is an important element of the pathogenesis of both alpha- and beta-HPVs, and the main difference between these two groups would be a mechanism employed to achieve the same goal. Beta-HPVs are defective for this zinc imbalance, an important growth-promoting function performed by E5 of alpha-HPVs, and inactivation of EVER proteins may compensate for the missing viral function . Indeed the transmembrane viral 16E5 and cellular EVER proteins interact both with the zinc transporter ZnT1, and are likely to modulate zinc homeostasis [115, 117]. The disruption of the cellular zinc-transporting complex achieved during the long co-evolution of HPV and the human species by two completely unrelated strategies underlines the general importance of the cellular zinc (im)balance in the papillomavirus life cycle and the central role played by E5.
In the absence of E5, beta-papillomaviruses have evolved a mechanism to escape immunosurveillance independent of E5. Indeed the E6 and E7 oncoproteins of cutaneous HPV-38 interfere with the interferon pathway. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation was associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1 . Thus, at least for some (high risk?) beta papillomavirus other E5-unrelated mechanisms take part in the process of viral replication and avoidance of immune control.
HPV E5 and carcinogenesis
HPV E5 proteins are not thought to play a role in the later steps of malignant progression because in high-risk HPV infections that progress to cancer the viral DNA typically integrates into the host genome often resulting in the loss of the E2 and E5 genes . However in contrast to other high-risk HPVs, HPV-16 DNA can exist in integrated, episomal or integrated and episomal form in malignant lesions of the cervix. In one study about 60% of HPV-16-positive cervical cancer expressed the 16E5 protein  and recent reports suggest that these tumours with episomal HPV-16 may have a more aggressive behaviour (Venuti, unpublished data). Nevertheless, the fact that a substantial proportion of the tumours do not express E5 indicates that the protein is not essential for HPV-16 mediated tumour progression.
Although 16E5 may not contribute to malignant progression, there is evidence that if E5 expression proceeds beyond early lesional stages, keratinocyte differentiation and immunological removal of infected cells does not take place increasing the likelihood of subsequent oncogenic transformation.
The presence of an E5 gene in all HR HPVs  and the detection of E5 variants with higher codon usage in high grade lesions  point to an important role of E5 during PV-induced pathogenesis. This assumption is further strengthen by studies on transgenic animals: (a) Transgenic mice expressing only HPV-16 E6 and E7 oncogenes in the basal epithelium develop fewer tumours than those arising in transgenic mice expressing HPV-16 E5, E6 and E7 genes  (b) Mice transgenic for 16E5 (without HPV 16 E6 and E7) develop skin tumours with high frequency .
One or more of the demonstrated biological activities of 16E5 may be responsible for early post-infection events enhancing the probability of subsequent carcinogenesis. Indeed viral persistence is considered to be an important factor in the neoplastic progression of a premalignant lesion . The 16E5-induced down regulation of MCH I  and MHC II , resulting in the infected cell evading immunosurveillance, may increase the duration and size of the HPV infection raising the probability that some infected cells will become transformed. The expansion of infected cells may also be favoured by 16E5-induced DNA synthesis in differentiating keratinocytes  and by 16E5-enhanced ligand-dependent activation of EGF-R [56, 58–60]. Furthermore the ability of 16E5 to inhibit gap-junction-mediated intercellular communication via connexin 43 interaction may render the infected cells more insensitive to homeostatic growth control signals from adjacent, non-infected cells [83, 84]. 16E5 induction of cell fusion may represent another critical event in the early stage of HPV-associated cervical cancer [41–43]. Thus, HR HPVs E5 is involved in the very early stage of carcinogenesis by prolonging the life and expanding the pool of the infected cells from which cancer may arise as a stochastic event. Finally E5 can be also involved in negative selection of cells harboring episomal virus and in turn favoring the selection of cells with integrated virus. Episome loss, associated with induction of antiviral response genes, could be a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by type I IFN pathway . Recently this activation of the antiviral state was shown to be induced by 16E5 through the stimulation of IRF-1 and IFN-β . In this scenario cervical carcinogenesis requires not only HR HPV integration, but also loss of inhibitory (E2 expression) episomes by the E5-induced establishment of an antiviral state that may accelerate episomal clearance.
BPV E5 is the best characterized of the E5 proteins and has provided the blueprint for investigations into HPV E5. E5 is the major BPV oncoprotein, only 44 amino acid in BPV type 1 (BPV-1) and 42 in BPV type 4 (BPV-4) [124–127]. Both proteins can be divided into two distinct domain: an amino-terminal domain which makes up the majority of the protein and consists of strongly hydrophobic leucine-rich membrane-spanning amino acid residues with a single hydrophilic amino acid at position 17 (glutamine in BPV-1 E5 and asparagine in BPV-4 E5), and a short hydrophilic carboxyl-terminal domain[125, 128]. Both E5 proteins localize in the endomembrane compartments of the GA, ER and plasma membrane. It has been suggested for BPV-1 E5 that helix-helix hydrophobic contacts within the TM domain play a critical role in functional dimer assembly and that the cystein-containing motif functions as additional dimer stabilization [129–131].
Cell transformation by BPV E5
Despite their structural similarity, BPV-1 and BPV-4 E5 differ in the ways they achieve cell transformation, likely reflecting the different origin of the cells hosting the two viruses. BPV-1 infects fibroblasts and skin keratinocytes giving rise to fibropapillomas, whereas BPV-4 infects solely the epithelial cells of the mucous epithelium of the upper gastrointestinal tract . Thus, while expression of BPV-1 E5 by itself is sufficient to fully transform mouse and primary human fibroblast cells [133, 134], BPV-4 E5 contributes to the transformed phenotype of primary cells by conferring anchorage independent growth, growth in low serum, focus formation and increased cell motility but only in the presence of the other viral oncoproteins, resembling in this respect 16E5 (Table 1) [127, 135, 136]. For both proteins the hydrophobic domain, the hydrophilic residues at position 17 and the hydrophilic C-terminal tail are critical for their transforming activities [135, 137].
BPV-1 E5 induces tumourigenic cell transformation by strongly and specifically binding to its cellular target, Platelet Derived Growth Factor Receptor β receptor (PDGFβ-R) tyrosine kinase in a ligand-independent manner [138–141]. The PDGFβ-R is a transmembrane protein which is normally activated by binding of its ligand PDGF. BPV-1 E5 can bind as a dimer to two monomers of the PDGFβ-R [140, 142, 143], inducing receptor dimerisation which in turn causes trans-phosphyorylation of specific tyrosine kinase residues within the cytoplasmic domain of the receptor resulting in mitogenic signalling . Studies using PDGFβ-R kinase inhibitors show that maintenance of transformation by BPV-1 E5 requires sustained PDGFβ-R activation [145, 146].
BPV-1 E5 interacts with the PDGFβ-R via its transmembrane domain [141, 142, 147], unlike the natural ligand PDGF which binds the receptor via its ligand binding domain Extensive characterisation of E5 demonstrates that there are essentially four residues important for PDGFβ-R binding and activation. These are the transmembrane glutamine (Gln17) important for both dimerisation of E5 and for its interaction with PDGFβ-R; the aspartic acid (Asp 33) residue at the juxtamembrane region which provides a negative charge required for receptor interaction and two terminal cysteine residues (Cys37, Cys39) essential for homodimerisation of E5 [125, 137, 145, 148, 149]. Interestingly, these four residues are conserved among the E5 proteins of fibropapillomaviruses  whereas other residues within the transmembrane region are less well conserved. Mutagenesis studies have demonstrated that BPV-1 E5 can tolerate a surprising number of mutations and that the maintenance of the hydrophobic nature of E5 and the conserved residues is enough to confer transforming activity [151–155].
Mutational analyses of the receptor itself have identified key residues important for function. The PDGFβ-R is as single span type I transmembrane receptor with three domains: an amino terminal domain which binds its receptor, a short membrane spanning region and an intracellular kinase domain. Two residues appear to be important for E5 interaction and transformation activity: a threonine 513 in the transmembrane domain is required for H bonding with Gln17 residue of the E5 protein and lysine at 499 in the juxtamembrane region with Asp33 in the E5 protein [156–158]. An important feature of BPV-1 E5 mediated PDGFβ-R activation is that activation occurs independently of PDGF since E5 can constitutively activate PDGFβ-R deletion mutants that lack the extracellular ligand-binding domain [141, 159].
The interaction between BPV-1 E5 and PDGFβ-R is highly specific. At physiological levels, E5 is able to interact with PDGFβ-R but not with other tyrosine kinase receptors including insulin receptors, basic fibroblast growth factor receptor or insulin-like growth factor receptors, however at higher levels E5 can interact with additional receptors [140, 144]. E5 cannot bind the PDGF α receptor, a closely related receptor to the PDGFβ-R whilst both of these receptors can be activated by PDGF [140–144]. The specificity is thought to be conferred by the transmembrane domain .
Most of the studies on BPV E5 and PDGFβ-R activation have been performed in vitro. Borzacchiello et al (2006) have shown that BPV-2 E5 interacts with and activates the PDGFβ-R in vivo in bovine urinary bladder cancers (Figure 3). Moreover, the binding of E5 to PDGFβ-R induces the activation of different signal transduction pathways: PDGFβ-R and phosphoinositide 3 kinase (PI3K) physically interact as do PDGFβ-R and the Grb2-Sos complex. PI3K-Akt and Grb2-Sos-Ras signals are all potentiated in cancer, but, as in in vitro models, the levels of Erk and Mek proteins are not significantly overexpressed [160–162] (Figure 4). BPV E5 is able to activate c-src, a non receptor tyr kinase, but not the closely related c-Fyn and PI3K . The activation of the latter is necessary but not sufficient to induce cell transformation independently of PDGFβ-R signalling [163, 164]. Unlike BPV-1 E5, BPV-4 E5 induces transformation of established cells independently from the constitutive activation of tyrosine kinase growth receptors (Table 1) . Cell transformation by BPV-4 E5 (either of established cells by itself or of primary cells in co-operation with other viral oncoproteins) is achieved through transactivation of the cyclin A gene promoter, increased cyclin A expression and cyclin A-associated kinase activity, and inhibition of the negative regulator of cell cycle, p27Kip1[135, 166–168]. Mutational analyses of BPV-4 E5 have demonstrate that, as is the case for BPV-1 E5, the residue at position 17 and the hydrophilic C-terminal tail are critical for its transforming activities . Thus both mutation of Asp 17 and deletion of the C-terminus hydrophilic domain completely abrogate cell transformation and E5 ability to activate the cyclin A pathway .
Down-regulation of MHC expression
The down-regulation of surface MHC I and retention of the MHC I complex in the GA were first shown in cells expressing BPV-4 E5  and then confirmed for other E5 proteins both from BPV and HPV. BPV E5 interferes with MHC I biosynthesis at multiple steps: transcriptional inhibition of the MHC class I heavy chain gene, degradation of the heavy chain and physical interaction with any residual heavy chain [169, 170]. Only the physical interaction with heavy chain is shared with 16E5 . Moreover, in the case of BPV-4 E5 the down-regulation of surface MHC I is irreversible, as interferon cannot restore the transport of the MHC I complex to the cell surface , while this is not the case for 16E5. This may reflect the fact that E5 is a "stronger" protein in BPV than in HPV.
In any event, the finding of MHC down-regulation by E5 has proved of great importance since it defined a new biological activity shared by both BPV and HPV E5 (Table 1), which allows the infected cell not to be effectively recognised by cytotoxic CTL .
BPV E5: other biological activities
BPV E5 interacts with the 16 kDa protein, a component of the V-ATPase [147, 171]. In the case of BPV-1 E5, this protein association exists as a tri-component complex, which contains also PDGFβ-R molecules. Mutational analyses have demonstrated that glutamic acid 143 in the transmembrane domain of the 16 kDa protein and glutamine 17 in the transmembrane domain of BPV-1 E5 are essential for E5-16 kDa stable association . The binding of the E5 with 16 kDa perturbs the correct assembly of V-ATPase and H+ pumping, with persistent alkalinization of the GA and endosomal vesicles  and may lead, directly or indirectly, to a marked loss of cell-cell communication through down-regulation of gap junctions (Table 1) [101, 173], rendering the transformed cells more insensitive to homeostatic growth control signals from adjacent normal cells. However, in the case of HPV-16 inhibition of gap junction intercellular communication has been attributed to the interaction of E5 with connexin 43 . E5-expressing bovine urinary bladder tumours show activated calpain 3, suggesting a possible involvement of this protein in urothelial carcinogenesis .
Targeting HPV-16 E5 for cervical treatment
16E5 activates many different cellular pathways involved mostly in the early stage of cervical carcinogenesis . Different studies have pointed out the possibility of E5 targeting for CaCx therapy. In animal studies, 16E5 delivered by an adenovirus vector reduces the growth of tumours and the E5 vaccine induces protection against tumours through CD8+ cytotoxic T cells (CTLs) . The same research group has further demonstrated that the stimulating epitope was an E5 peptide (aminoacids 25-33) and vaccination with this peptide carrying CpG oligonucleotides reduced tumour growth . 16E5 modulates different cellular pathways and the targeting of these relevant pathways may lead in a near future to a possible therapeutic approach. In addition, E5 is more consistently expressed in the early stage of viral infection and in precancerous lesions , and therefore E5 or E5 altered pathways could be targeted to cure the infection and to prevent precancerous lesions from progressing into invasive cancers. 16E5 interacts with many different pathways including upregulation of growth factor signalling, induction of inflammatory cell signalling, angiogenesis and antiapoptosis (Figure 2). Furthermore E5 participates in malignant transformation by supplementing (added value) the roles of E6 and E7. All these E5-induced signalling pathways (Figure 2) or E5 itself can be addressed by therapies already utilised against the other HPV oncogenes such as radioimmunotherapy, oncolytic adenoviruses , gene silencing using short interfering RNA (siRNA)  and others, which have been originally used to target the E5-upregulated pathways. EGF-R, COX-2 or ET1 inhibitors should be useful in controlling E5 activity in the precancerous lesions as well as other small molecules interfering with downstream molecules. Finally, E5 may be considered a viroporin, like the VP4 of SV40  and, therefore, susceptible of therapy by compounds that affect similar structures produced by other viruses. Indeed amantidine, amiloride, long-alkyl-chain iminosugar derivatives and new compounds seem to affect, to various extent, the activity of the 16E5 protein in vitro (A. Macdonald, personal communication, 11 DNA TV Meeting, Trieste, Italy, 2011)
The main activities of E5 can be resumed as follows:
Although E6 and E7 provide the primary transforming activities of HR HPVs, E5 can augment their function and contribute to tumour progression:
When expressed alone, HPV E5 has weak transforming activity.
In transgenic mouse models, 16E5 expression in the skin produces epithelial hyperproliferation with spontaneous tumour formation, whereas in estrogen-treated mice, expression of E5 alone can induce cancers , suggesting a role for E5 as a true oncoprotein.
The presence in HPV 16-positive cervical tumours of viral episomes in addition to viral integrants leads to the hypothesis that there are multiple pathways to HPV induced tumourigenesis see above. Beside the basic hypothesis of high-level expression of E6 and E7 consequent to the abrogation of the repressive effects of E2 due to the loss of the E2 gene during integration, another pathway can be active in cells that still maintain viral episomes, in which E5 would augment the activity of E6 and E7. The expression of E5 is increased on differentiation to promote proliferation of differentiated cells and productive viral replication.
The localization of HPV E5 to the endoplasmic reticulum suggests its activity may be related to the trafficking of cytoplasmic membrane proteins through this cellular compartment, in particular of growth factor receptors and of molecules involved in immune control.
There are multiple documented intracellular binding targets for 16E5 such as the EGF receptor family member ErbB4 , the 16-kDa subunit of the vacuolar H+-ATPase [39, 55], the heavy chain of HLA type I , calnexin , the zinc transporter ZnT-1, the EVER1 and EVER2 transmembrane channel-like proteins [115, 117] the nuclear import receptor family member KNβ3 , BAP31 and A4 [44, 52] (Figure 2).
However the role of 16E5 in carcinogenesis seems to be limited to the early stages of cervical carcinogenesis because the E5 gene is frequently deleted when the HPV genome is integrated during malignant progression [28, 68, 183]. Nevertheless, the expression of 16E5 as detected by immunohistochemistry, was reported in approximately 80, 90 and 60% of HPV 16-infected LSILs, high-grade SILs and cervical carcinomas, respectively . Furthermore data from a limited number of patients were presented at the 1st International Workshop on E5 Oncogene  suggesting that E5 expression may lead to a worse response to treatment with taxanes (Venuti, unpublished data). Therefore, targeting E5 which is frequently expressed in earlier stages of malignant transformation may be a rational approach for preventing premalignant lesions from progressing into invasive cervical cancers . The recent successful eradication of HPV-induced tumours in mice by anti-E5 vaccination  indicates that E5 can be used in immunotherapy. The neutralization of E5 or of E5-induced signalling pathways, therefore, whether immunological or chemical, could be advantageous particularly in HPV infections and pre-cancerous lesions where there are no treatments available.
cancer of the uterine cervix
- PAP test:
late structural proteins
open reading frame
low grade squamous intraepithelial lesions
cervical intraepithelial neoplasia
- MHC I:
Major histocompatibility complex class I
- MHC II:
Major histocompatibility complex class II
human leukocyte antigen
cytotoxic T Lymphocyte
epidermal growth factor receptor
mitogen-activated protein kinase
protein kinase C
cyclin-dependent protein kinase inhibitors
Tumor necrosis factor-related apoptosis-inducing ligand
Death-Inducing Signalling Complex
Tumor necrosis factor ligand superfamily member
Prostaglandin E2 receptor
X-box-binding protein 1
Serine/threonine-protein kinase/endoribonuclease IRE1
Cyclic AMP-responsive element-binding protein
Platelet Derived Growth Factor Receptor β subunit receptor
IARC Working Group: Human papillomaviruses. 1995, 64: Lyon, International Agency for research on cancer,
Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999, 189: 12-19.
Frazer IH: Prevention of cervical cancer through papillomavirus vaccination. Nat Rev Immunol. 2004, 4: 46-54.
Parkin DM, Bray F: Chapter 2: The burden of HPV-related cancers. Vaccine. 2006, 24: 11-25.
Syrjänen S, Lodi G, von Bültzingslöwen I, Aliko A, Arduino P, Campisi G, Challacombe S, Ficarra G, Flaitz C, Zhou HM, Maeda H, Miller C, Jontell M: Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review. Oral Dis. 2011, 17: 58-72.
Klozar J, Tachezy R, Rotnáglová E, Koslabová E, Saláková M, Hamsíková E: Human papillomavirus in head and neck tumors: epidemiological, molecular and clinical aspects. Wien Med Wochenschr. 2010, 160: 305-309.
Zafer E, Ergun MA, Alver G, Sahin FI, Yavuzer S, Ekmekci A: Detection and typing of human papillomavirus in non-small cell lung cancer. Respiration. 2004, 71: 88-90.
Gillison ML: Human papillomavirus-associated head and neck cancer is a distinct epidemiologic, clinical, and molecular entity. Semin Oncol. 2004, 31: 744-754.
Syrjänen KJ: HPV infections in benign and malignant sinonasal lesions. J Clin Pathol. 2003, 56: 174-181.
zur Hausen H: Yohei Ito Memorial Lecture: Papillomaviruses in human cancers. Leukemia. 1999, 13: 1-5.
Audeau A, Han HW, Johnston MJ, Whitehead MW, Frizelle FA: Does human papilloma virus have a role in squamous cell carcinoma of the colon and upper rectum?. Eur J Surg Oncol. 2002, 28: 657-660.
Damin AP, Karam R, Zettler CG, Caleffi M, Alexandre CO: Evidence for an association of human papillomavirus and breast carcinomas. Breast Cancer Res Treat. 2004, 84: 131-137.
Shigehara K, Sasagawa T, Kawaguchi S, Nakashima T, Shimamura M, Maeda Y, Konaka H, Mizokami A, Koh E, Namiki M: Etiologic role of human papillomavirus infection in bladder carcinoma. Cancer. 2011, 117: 2067-2076.
Martinez-Fierro ML, Leach RJ, Gomez-Guerra LS, Garza-Guajardo R, Johnson-Pais T, Beuten J, Morales-Rodriguez IB, Hernandez-Ordoñez MA, Calderon-Cardenas G, Ortiz-Lopez R, Rivas-Estilla AM, Ancer-Rodriguez J, Rojas-Martinez A: Identification of viral infections in the prostate and evaluation of their association with cancer. BMC Cancer. 2010, 10: 326-
Borzacchiello G, Roperto F, Nasir L, Campo MS: Human papillomavirus research: do we still need animal models?. Int J Cancer. 2009, 125: 739-740.
Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990, 248: 76-79.
Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM: The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990, 63: 1129-1136.
Moody CA, Laimins LA: Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010, 10: 550-560.
Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen H, de Villiers EM: Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology. 2010, 401: 70-79.
Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ, : Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003, 348: 518-527.
Nath R, Mant CA, Kell B, Cason J, Bible JM: Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts. Cancer Cell Int. 2006, 6: 19-27.
Bravo IG, Alonso A: Mucosal human papillomaviruses encode four different E5 proteins whose chemistry and phylogeny correlate with malignant or benign growth. J Virol. 2004, 78: 13613-13626.
Schiffman M, Herrero R, Desalle R, Hildesheim A, Wacholder S, Rodriguez AC, Bratti MC, Sherman ME, Morales J, Guillen D, Alfaro M, Hutchinson M, Wright TC, Solomon D, Chen Z, Schussler J, Castle PE, Burk RD: The carcinogenicity of human papillomavirus types reflects viral evolution. Virology. 2005, 337: 76-84.
Garcia-Vallve S, Alonso A, Bravo IG: Papillomaviruses: different genes have different histories. Trends Microbiol. 2005, 13: 514-521.
Longworth MS, Laimins LA: Pathogenesis of human papillomaviruses in differentiating epithelia. Microbiol Mol Biol Rev. 2004, 68: 362-372.
Burnett S, Jareborg N, DiMaio D: Localization of bovine papillomavirus type 1 E5 protein to transformed basal keratinocytes and permissive differentiated cells in fibropapilloma tissue. Proc Natl Acad Sci USA. 1992, 89: 5665-5669.
Araibi EH, Marchetti B, Ashrafi GH, Campo MS: Downregulation of major histocompatibility complex class I in bovine papillomas. J Gen Virol. 2004, 85: 2809-2814.
Chang JL, Tsao YP, Liu DW, Huang SJ, Lee WH, Chen SL: The expression of HPV- 16 E5 protein in squamous neoplastic changes in the uterine cervix. J Biomed Sci. 2001, 8: 206-213.
Chen SL, Mounts P: Transforming activity of E5a protein of human papillomavirus type 6 in NIH 3T3 and C127 cells. J Virol. 1990, 64: 3226-3233.
Leechanachai P, Banks L, Moreau F, Matlashewski G: The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene. 1992, 7: 19-25.
Leptak C, Ramon y Cajal S, Kulke R, Horwitz BH, Riese DJ, Dotto GP, DiMaio D: Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16. J Virol. 1991, 65: 7078-7083.
Pim D, Collins M, Banks L: Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor. Oncogene. 1992, 7: 27-32.
Bouvard V, Matlashewski G, Gu ZM, Storey A, Banks L: The human papillomavirus type 16 E5 gene cooperates with the E7 gene to stimulate proliferation of primary cells and increases viral gene expression. Virology. 1994, 203: 73-80.
Straight SW, Herman B, McCance DJ: The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes. J Virol. 1995, 69: 3185-3192.
Valle FG, Banks L: The human papillomavirus (HPV)-6 and HPV-16 E5 proteins co-operate with HPV-16 E7 in the transformation of primary rodent cells. J Gen Virol. 1995, 76: 1239-1245.
Venuti A, Salani D, Poggiali F, Manni V, Bagnato A: The E5 oncoprotein of human papillomavirus type 16 enhances endothelin-1-induced keratinocyte growth. Virology. 1998, 248: 1-5.
Ghai J, Ostrow RS, Tolar J, McGlennen RC, Lemke TD, Tobolt D, Liu Z, Faras AJ: The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells. Proc Natl Acad Sci USA. 1996, 93: 12879-12884.
Rho J, de Villiers EM, Choe J: Transforming activities of human papillomavirus type 59 E5, E6 and E7 open reading frames in mouse C127 cells. Virus Res. 1996, 44: 57-65.
Conrad M, Bubb VJ, Schlegel R: The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein. J Virol. 1993, 67: 6170-6178.
Disbrow GL, Sunitha I, Baker CC, Hanover J, Schlegel R: Codon optimization of the HPV-16 E5 gene enhances protein expression. Virology. 2003, 311: 105-114.
Hu L, Ceresa BP: Characterization of the plasma membrane localization and orientation of HPV16 E5 for cell-cell fusion. Virology. 2009, 393: 135-143.
Hu L, Plafker K, Vorozhko V: Human papillomavirus E5 induces bi-nucleated cell formation by cell-cell fusion. Virology. 2009, 384: 125-134.
Hu L, Potapova TA, Li S: Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication. Virology. 2010, 405: 342-351.
Krawczyk E, Suprynowicz FA, Sudarshan SR, Schlegel R: Membrane orientation of the human papillomavirus type 16 E5 oncoprotein. J Virol. 2010, 84: 1696-1703.
Hanover JA, Schlegel R, Suprynowicz FA: Karyopherin β3: a new cellular target for the HPV-16 E5 oncoprotein. Biochem Biophys Res Commun. 2008, 371: 684-688.
Deane R, Schäfer W, Zimmermann HP, Mueller L, Goörlich D, Prehn S, Ponstingl H, Bischoff FR: Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1. Mol Cell Biol. 1997, 17: 5087-5096.
Yaseen NR, Blobel G: Cloning and characterization of human karyopherin β3. Proc Natl Acad Sci USA. 1997, 94: 4451-4456.
Burkhardt A, Willingham M, Gay C, Jeang KT, Schlegel R: The E5 oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasma membranes. Virology. 1989, 170: 334-339.
Yang DH, Wildeman AG, Sharom FJ: Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16. Protein Expr Purif. 2003, 30: 1-10.
Gieswein CE, Sharom FJ, Wildeman AG: Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions. Virology. 2003, 313: 415-426.
Gruener M, Bravo IG, Momburg F, Alonso A, Tomakidi P: The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin expressing but not in calnexin-deficient cells. Virol J. 2007, 4: 116-130.
Regan JA, Laimins LA: Bap31 is a novel target of the human papillomavirus E5 protein. J Virol. 2008, 82: 10042-10051.
Cortese MS, Ashrafi GH, Campo MS: All four di-leucine motifs in the first hydrophobic domain of the E5 oncoprotein of human papillomavirus type 16 are essential for surface MHC class I downregulation activity and E5 endomembrane localization. Int J Cancer. 2010, 126: 1675-1682.
Adam JL, Briggs MW, McCance DJ: A mutagenic analysis of the E5 protein of human papillomavirus type 16 reveals that E5 binding to the vacuolar H+-ATPase is not sufficient for biological activity, using mammalian and yeast expression systems. Virology. 2000, 272: 315-325.
Rodriguez MI, Finbow ME, Alonso A: Binding of human papillomavirus 16 E5 to the 16 kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation. Oncogene. 2000, 19: 3727-3732.
Krawczyk E, Suprynowicz FA, Liu X, Dai Y, Hartmann DP, Hanover J, Schlegel R: Koilocytosis: a cooperative interaction between the human papillomavirus E5 and E6 oncoproteins. Am J Pathol. 2008, 173: 682-688.
Krawczyk E, Suprynowicz FA, Hebert JD, Kamonjoh CM, Schlegel R: The human papillomavirus type 16 E5 oncoprotein translocates calpactin I to the perinuclear region. J Virol. 2011, 85: 10968-10975.
Straight SW, Hinkle PM, Jewers RJ, McCance DJ: The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes. J Virol. 1993, 67: 4521-4532.
Mittal KR, Chan W, Demopoulos RL: Sensitivity and specificity of various morphological features of cervical condylomas. Arch Pathol Lab Med. 1990, 114: 1038-1041.
Prasad CJ, Sheets E, Selig AM, McArthur MC, Crum CP: The bi-nucleated squamous cell: histologic spectrum and relationship to low-grade squamous intraepithelial lesions. Mod Pathol. 1993, 6: 313-317.
Wright TC, Gatscha RM, Luff RD, Prey MU: Epithelial cell abnormalities: squamous. The Bethesda System for Reporting Cervical Cytology. Edited by: Solomon D, Nayar R. 2004, 89-123. New York: Springer-Verlag, 2,
Olaharski AJ, Sotelo R, Solorza-Luna G, Gonsebatt ME, Guzman P, Mohar A, Eastmond DA: Tetraploidy and chromosomal instability are early events during cervical carcinogenesis. Carcinogenesis. 2006, 27: 337-343.
Egawa K: Do human papillomaviruses target epidermal stem cells?. Dermatology. 2003, 207: 251-254.
McMurray HR, Nguyen D, Westbrook TF, McAnce DJ: Biology of human papillomaviruses. Int J Exp Pathol. 2001, 82: 15-33.
zur Hausen H: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002, 2: 342-350.
Fehrmann F, Laimins LA: Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene. 2003, 22: 5201-5207.
Genther Williams SM, Disbrow GL, Schlegel R, Lee D, Threadgill DW, Lambert PF: Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene. Cancer Research. 2005, 65: 6534-6542.
DiMaio D, Mattoon D: Mechanism s of cell transformation by papillomavirus E5 proteins. Oncogene. 2001, 20: 7866-7873.
Thomsen P, van Deurs B, Norrild B, Kayser L: The HPV16 E5 oncogene inhibits endocytic trafficking. Oncogene. 2000, 19: 6023-6032.
Crusius K, Rodriguez I, Alonso A: The human papillomavirus type 16 E5 protein modulates ERK1/2 and p38 MAP kinase activation by an EGFR-independent process in stressed human keratinocytes. Virus Genes. 2000, 20: 65-69.
Crusius K, Auvinen E, Alonso A: Enhancement of EGF- and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein. Oncogene. 1997, 15: 1437-1444.
Gu Z, Matlashewski G: Effect of human papillomavirus type 16 oncogenes on MAP kinase activity. J Virol. 1995, 69: 8051-8056.
Chen SL, Huang CH, Tsai TC, Lu KY, Tsao YP: The regulation mechanism of c-jun and junB by human papillomavirus type 16 E5 oncoprotein. Arch Virol. 1996, 141: 791-800.
Chen SL, Lin YK, Li LY, Tsao YP, Lo HY, Wang WB, Tsai TC: E5 proteins of human papillomavirus types 11 and 16 transactivate the c-fos promoter through the NF1 binding element. J Virol. 1996, 70: 8558-8563.
Crusius K, Auvinen E, Steuer B, Gaissert H, Alonso A: The human papillomavirus type 16 E5-protein modulates ligand-dependent activation of the EGF receptor family in the human epithelial cell line HaCaT. Exp Cell Res. 1998, 241: 76-83.
Hwang ES, Nottoli T, Dimaio D: The HPV16 E5 protein: expression, detection, and stable complex formation with transmembrane proteins in COS cells. Virology. 1995, 211: 227-233.
Chen SL, Lin ST, Tsai TC, Hsiao WC, Tsao YP: ErbB4 (JM-b/CYT-1)-induced expression and phosphorylation of c-Jun is abrogated by human papillomavirus type 16 E5 protein. Oncogene. 2007, 26: 42-53.
Simeone P, Teson M, Latini A, Carducci M, Venuti A: Endothelin-1 could be one of the targets of psoriasis therapy. Br J Dermatol. 2004, 151: 1273-1275.
Bagnato A, Loizidou M, Pflug BR, Curwen J, Growcott J: Role of the endothelin axis and its antagonists in the treatment of cancer. Br J Pharmacol. 2011, 163: 220-233.
Belleudi F, Leone L, Purpura V, Cannella F, Scrofani C, Torrisi MR: HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signalling and endocytic traffic. Oncogene. 2011,
Tsao YP, Li LY, Tsai TC, Chen SL: Human papillomavirus type 11 and 16 E5 represses p21(WafI/SdiI/CipI) gene expression in fibroblasts and keratinocytes. J Virol. 1996, 70: 7535-7539.
Pedroza-Saavedra A, Lam EW, Esquivel-Guadarrama F, Gutierrez-Xicotencatl L: The human papillomavirus type 16 E5 oncoprotein synergizes with EGFreceptor signalling to enhance cell cycle progression and the down-regulation of p27(Kip1). Virology. 2010, 400: 44-52.
Oelze I, Kartenbeck J, Crusius K, Alonso A: Human papillomavirus type 16 E5 protein affects cell-cell communication in an epithelial cell line. J Virol. 1995, 69: 4489-4494.
Tomakidi P, Cheng H, Kohl A, Komposch G, Alonso A: Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein. Cell Tissue Res. 2000, 301: 323-327.
Stoler MH, Rhodes CR, Whitbeck A, Wolinsky SM, Chow LT, Broker TR: Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol. 1992, 23: 117-128.
Kell B, Jewers RJ, Cason J, Pakarian F, Kaye JN, Best JM: Detection of E5 oncoprotein in human papillomavirus type 16-positive cervical scrapes using antibodies raised to synthetic peptides. J Gen Virol. 1994, 75: 2451-2456.
Kabsch K, Alonso A: The human papillomavirus type 16 E5 protein impairs TRAIL- and FasL-mediated apoptosis in HaCaT cells by different mechanisms. J Virol. 2002, 76: 12162-12172.
Kabsch K, Mossadegh N, . Kohl A, Komposch G, Schenkel J, Alonso A, Tomakidi P: The HPV-16 E5 protein inhibits TRAIL- and FasL-mediated apoptosis in human keratinocyte raft cultures. Intervirology. 2004, 47: 48-56.
Zhang B, Spandau DF, Roman A: E5 protein of human papillomavirus type 16 protects human foreskin keratinocytes from UV B-irradiation-induced apoptosis. J Virol. 2002, 76: 220-231.
Kabsch K, Alonso A: The human papillomavirus type 16 (HPV-16) E5 protein sensitizes human keratinocytes to apoptosis induced by osmotic stress. Oncogene. 2002, 21: 947-953.
Tardif KD, Mori K, Kaufman RJ, Siddiqui A: Hepatitis C virus suppresses the IRE1-XBP1 pathway of the unfolded protein response. J Biol Chem. 2004, 279: 17158-17164.
Schiffman M, Castle PE: The promise of global cervical-cancer prevention. N Engl J Med. 2005, 353: 2101-2104.
Condjella R, Liu X, Suprynowicz F, Yuan H, Sudarshan S, Dai Y, Schlegel R: The canine papillomavirus E5 protein signals from the endoplasmic reticulum. J Virol. 2009, 83: 12833-12841.
Wu R, Abramson AL, Shikowitz MJ, Dannenberg AJ, Steinberg BM: Epidermal growth factor-induced cyclooxygenase-2 expression is mediated through phosphatidylinositol-3 kinase, not mitogen-activated protein/extracellular signal-regulated kinase kinase, in recurrent respiratory papillomas. Clin Cancer Res. 2005, 11: 6155-6161.
Wu R, Coniglio SJ, Chan A, Symons MH, Steinberg BM: Up-regulation of Rac1 by epidermal growth factor mediates COX-2 expression in recurrent respiratory papillomas. Mol Med. 2007, 13: 143-150.
Subbaramaiah K, Dannenberg AJ: Cyclooxygenase-2 transcription is regulated by human papillomavirus 16 E6 and E7 oncoproteins: evidence of a corepressor/coactivator exchange. Cancer Res. 2007, 67: 3976-3985.
Kim SH, Oh JM, No JH, Bang YJ, Juhnn YS, Song YS: Involvement of NFkappaB and AP-1 in COX-2 upregulation by human papillomavirus 16 E5 oncoprotein. Carcinogenesis. 2009, 30: 753-757.
Sudarshan SR, Schlegel R, Liu X: The HPV-16 E5 protein represses expression of stress pathway genes XBP-1 and COX-2 in genital keratinocytes. Biochem Biophys Res Commun. 2010, 399: 617-622.
Oh JM, Lee YI, Song YS, Kim WH, Juhnn YS: Human papillomavirus E5 protein induces expression of the EP4 subtype of prostaglandin E2 receptor in cyclic AMP response element-dependent pathways in cervical cancer cells. Carcinogenesis. 2009, 30: 141-149.
Stanley MA, Pett MR, Coleman N: HPV: from infection to cancer. Biochem Soc Trans. 2007, 35: 1456-1460.
O'Brien PM, Campo MS: Evasion of host immunity directed by papillomavirus encoded proteins. Virus Res. 2002, 1: 103-118.
Ashrafi GH, Haghshenas MR, Marchetti B, O'Brien PM, Campo MS: The E5 protein of human papillomavirus type 16 selectively down-regulates surface HLA class. Int J Cancer. 2005, 113: 276-283.
Schapiro F, Sparkowski J, Adduci A, Schlegel R, Grinstein S: Golgi alkalinization by the papillomavirus E5 oncoprotein. J Cell Biol. 2000, 148: 305-315.
Ashrafi GH, Haghshenas M, Marchetti B, Campo MS: E5 protein of human papillomavirus 16 downregulates HLA class I and interacts with the heavy chain via its first hydrophobic domain. Int J Cancer. 2006, 119: 2105-2112.
Araibi EH, Marchetti B, Dornan ES, Ashrafi GH, Dobromylskyj M, Ellis SA, Campo MS: The E5 oncoprotein of BPV-4 does not interfere with the biosynthetic pathway of non-classical MHC class I. Virology. 2006, 353: 174-183.
Campo MS, Graham SV, Cortese MS, Ashrafi GH, Araibi EH, Dornan ES, Miners K, Nunes C, Man S: HPV-16 E5 down-regulates expression of surface HLA class I and reduces recognition by CD8 T cells. Virology. 2010, 407: 137-142.
Zhang B, Li P, Wang E, Brahmi Z, Dunn KW, Blum JS, Roman A: The E5 protein of human papillomavirus type 16 perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-γ. Virology. 2003, 310: 100-108.
Barnard P, Payne E, McMillan N: The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon α. Virology. 2000, 277: 411-419.
Lee SJ, Cho YS, Cho MC, Shim JH, Lee KA, Ko KK, Choe YK, Park SN, Hoshino T, Kim S, Dinarello CA, Yoon DY: Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells. J Immunol. 2001, 167: 497-504.
Ronco LV, Karpova AY, Vidal M, Howley PM: Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev. 1998, 12: 2061-2072.
Miura S, Kawana K, Schust DJ, Fujii T, Yokoyama T, Iwasawa Y, Nagamatsu T, Adachi K, Tomio A, Tomio K, Kojima S, Yasugi T, Kozuma S, Taketani Y: CD1d, a sentinel molecule bridging innate and adaptive immunity, is downregulated by the human papillomavirus (HPV) E5 protein: a possible mechanism for immune evasion by HPV. J Virol. 2010, 84: 11614-11623.
Obalek S, Favre M, Szymanczyk J, Misiewicz J, Jablonska S, Orth G: Human papillomavirus (HPV) types specific of epidermodysplasia verruciformis detected in warts induced by HPV3 or HPV3-related types in immunosuppressed patients. J Investig Dermatol. 1992, 98: 936-941.
Orth G: Genetics of epidermodysplasia verruciformis: insights into host defense against papillomaviruses. Semin Immunol. 2006, 18: 362-374.
Ramoz N, Rueda LA, Bouadjar B, Montoya LS, Orth G, Favre M: Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis. Nat Genet. 2002, 32: 579-581.
Lazarczyk M, Pons C, Mendoza JA, Cassonnet P, Jacob Y, Favre M: Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses. J Exp Med. 2008, 205: 35-42.
Palmiter RD, Findley SD: Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 1995, 14: 639-649.
Orth G: Host defenses against human papillomaviruses: lessons from epidermodysplasia verruciformis. Curr Top Microbiol Immunol. 2008, 321: 59-83.
Cordano P, Gillan V, Bratlie S, Bouvard V, Banks L, Tommasino M, Campo MS: The E6 E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway. Virology. 2008, 377: 408-418.
Bible JM, Mant C, Best JM, Kell B, Starkey WG, Shanti Raju K, Seed P, Biswas C, Muir P, Banatvala JE, Cason J: Cervical lesions are associated with human papillomavirus type 16 intratypic variants that have high transcriptional activity and increased usage of common mammalian codons. J Gen Virol. 2000, 81: 1517-1527.
Riley RR, Duensing S, Brake T, Münger K, Lambert PF, Arbeit JM: Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis. Cancer Res. 2003, 63: 4862-4871.
Genther SM, Sterling S, Duensing S, Münger K, Sattler C, Lambert PF: Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J Virol. 2003, 77: 2832-2842.
Pett MR, Herdman MT, Palmer RD, Yeo GS, Shivji MK, Stanley MA, Coleman N: Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response. Proc Natl Acad Sci USA. 2006, 103: 3822-3827.
Muto V, Stellacci E, Lamberti AG, Perrotti E, Carrabba A, Matera G, Sgarbanti M, Battistini A, Liberto MC, Focà A: Human papillomavirus type 16 E5 protein induces expression of beta interferon through interferon regulatory factor 1 in human keratinocytes. J Virol. 2011, 85: 5070-5080.
Schiller JT, Vass WC, Vousdan KH, Lowy DR: The E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene. J Virol. 1986, 57: 1-6.
Schlegel R, Wade-Glass M, Rabson MS, Yang YC: The E5 transforming gene of bovine papillomavirus encodes a small hydrophobic polypeptide. Science. 1986, 233: 464-467.
Burkhardt A, DiMaio D, Schlegel R: Genetic and biochemical definition of the bovine papillomavirus E5 transforming protein. EMBO J. 1987, 8: 2381-2385.
Pennie WD, Grindlay GJ, Cairney M, Campo MS: Analysis of the transforming functions of bovine papillomavirus type 4. Virology. 1993, 19: 614-620.
Marchetti B, Ashrafi GH, Tsirimonaki E, O'Brien PM, Campo MS: The bovine papillomavirus oncoprotein E5 retains MHC class I molecole in the Golgi apparatus and prevents their transport to the cell surface. Oncogene. 2002, 21: 7808-7816.
King G, Oates J, Patel D, van den Berg HA, Dixon AM: Towards a structural understanding of the smallest known oncoprotein: Investigation of the bovine papillomavirus E5 protein using solution-state NMR. Biochim Biophys Acta. 2011, 1808: 1943-1501.
Windisch D, Hoffmann S, Afonin S, Vollmer S, Benamira S, Langer B, Bürck J, Muhle-Goll C, Ulrich AS: Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5. Biophys J. 2010, 99: 1764-1772.
Oates J, Hicks M, Dafforn TR, DiMaio D, Dixon AM: In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain. Biochemistry. 2008, 47: 8985-8992.
Campo MS, Moar MH, Jarret WFH, Laird HM: A new papillomavirus associated with alimentary cancer in cattle. Nature. 1980, 286: 180-182.
Bergman P, Ustav M, Sedman J, Moreno-Lopéz J, Vennström B, Pettersson U: The E5 gene of bovine papillomavirus type 1 is sufficient for complete oncogenic transformation of mouse fibroblasts. Oncogene. 1988, 2: 453-459.
Petti LM, Ray FA: Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein. Cell growth differ. 2000, 11: 395-408.
O'Brien V, Ashrafi GH, Grindlay GJ, Anderson R, Campo MS: A Mutational Analysis of the Transforming Functions of the E8 Protein of Bovine Papillomavirus Type 4. Virology. 1999, 255: 385-394.
Tsirimonaki E, Ullah R, Marchetti B, Ashrafi GH, McGarry L, Ozanne B, Campo MS: Similarities and differences between the E5 oncoproteins of bovine papillomaviruses type 1 and type 4: cytoskeleton, motility and invasiveness in E5-transformed bovine and mouse cells. Virus Res. 2006, 115: 158-168.
Horwitz BH, Settleman J, Prakash SS, DiMaio D: Structure, activity, and regulation of the bovine papillomavirus E5 gene and its transforming protein product. Curr Top Microbiol Immunol. 1989, 144: 143-151.
Petti L, Nilson LA, DiMaio D: Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein. EMBO J. 1991, 10: 845-855.
Nilson LA, DiMaio D: Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein. Mol Cell Biol. 1993, 13: 4137-4145.
Goldstein DJ, Li W, Wang LM: The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation. J Virol. 1994, 68: 4432-4441.
Drummond-Barbosa DA, Vaillancourt RR, Kazlauskas A, DiMaio D: Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signalling. Mol Cell Biol. 1995, 15: 2570-2581.
Cohen BD, Goldstein DJ, Rutledge L, Vass WC, Lowy DR, Schlegel R, Schiller JT: Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain. J Virol. 1993, 67: 5303-5311.
Petti L, DiMaio D: Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells. Proc Natl Acad Sci USA. 1992, 89: 6736-6740.
Petti L, DiMaio D: Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells. J Virol. 1994, 68: 3582-3592.
Klein O, Kegler-Ebo D, Su J, Smith S, DiMaio D: The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells. J Virol. 1999, 73: 3264-3272.
Lai CC, Edwards AP, DiMaio D: Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor. J Virol. 2005, 79: 1924-1929.
Goldstein DJ, Andresson T, Sparkowski JJ, Schlegel R: The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions. EMBO J. 1992, 11: 4851-4859.
Klein O, Polack GW, Surti T, Kegler-Ebo D, Smith SO, DiMaio D: Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation. J Virol. 1998, 72: 8921-8932.
Surti T, Klein O, Aschheim K, DiMaio D, Smith SO: Structural models of the bovine papillomavirus E5 protein. Proteins. 1998, 33: 601-612.
Kulke R, DiMaio D: Biological properties of the deer papillomavirus E5 gene in mouse C127 cells: growth transformation, induction of DNA synthesis, and activation of the platelet-derived growth factor receptor. J Virol. 1991, 65: 4943-4949.
Horwitz BH, Burkhardt AL, Schlegel R, DiMaio D: 44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids. Mol Cell Biol. 1988, 8: 4071-4078.
Horwitz BH, Weinstat DL, DiMaio D: Transforming activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids. J Virol. 1989, 63: 4515-4519.
Meyer AN, Xu Y-F, Webster MK, Smith AE, Donoghue DJ: Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein. Proc Natl Acad Sci USA. 1994, 91: 4634-4638.
Kulke R, Horwitz BH, Zibello T, DiMaio D: The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine. J Virol. 1992, 66: 505-511.
Mattoon D, Gupta K, Doyon J, Loll PJ, DiMaio D: Identification of the transmembrane dimer interface of the bovine papillomavirus E5 protein. Oncogene. 2001, 20: 3824-3834.
Nappi VM, Petti LM: Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor. J Virol. 2002, 76: 7976-7986.
Nappi VM, Schaefer JA, Petti LM: Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein. J Biol Chem. 2002, 277: 47149-47159.
Petti LM, Reddy V, Smith SO, DiMaio D: Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein. J Virol. 1997, 71: 7318-7327.
Staebler A, Pierce JH, Brazinski S Heidaran MA, Li W, Schlegel R, Goldstein DJ: Mutational analysis of the beta-type platelet-derived growth factor receptor defines the site of interaction with the bovine papillomavirus type 1 E5 transforming protein. J Virol. 1995, 69: 6507-6517.
Corteggio A, Urraro C, Roperto S, Roperto F, Borzacchiello G: Phosphatidylinositol-3-kinase-AKT pathway, phospho-JUN and phospho-JNK expression in spontaneously arising bovine urinary bladder tumours. J Comp Pathol. 2010, 143: 173-178.
Corteggio A, Di Geronimo O, Roperto S, Roperto F, Borzacchiello G: Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas. Vet J. 2011,
Petti LM, Ricciardi EC, Page HJ, Porter KA: Transforming signals resulting from sustained activation of the PDGFbeta receptor in mortal human fibroblasts. J Cell Sci. 2008, 121: 1172-1182.
Suprynowicz FA, Baege A, Sunitha I, Schlegel R: c-Src activation by the E5 oncoprotein enables transformation independently of PDGF receptor activation. Oncogene. 2002, 21: 1695-1706.
Suprynowicz FA, Sparkowski J, Baege A, Schlegel R: E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation. J Biol Chem. 2000, 275: 5111-5119.
Grindlay GJ, Campo MS, O'Brien V: Transactivation of the cyclin A promoter by bovine papillomavirus type 4 E5 protein. Virus Res. 2005, 108: 29-38.
O'Brien V, Campo MS: BPV-4 E8 transforms NIH3T3 cells, up-regulates cyclin A and cyclin A-associated kinase activity and de-regulates expression of the cdk inhibitor p27Kip1. Oncogene. 1998, 17: 293-301.
O'Brien V, Grindlay GJ, Campo MS: Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes. J Biol Chem. 2001, 276: 33861-33868.
Zago M, Campo MS, O'Brien V: Cyclin A expression and growth in suspension can be uncoupled from p27 deregulation and extracellular signal-regulated kinase activity in cells transformed by bovine papillomavirus type 4 E5. J Gen Virol. 2004, 85: 3585-3595.
Ashrafi GH, Tsirimonaki E, Marchetti B, O'Brien PM, Sibbet GJ, Andrew L, Campo MS: Down-regulation of MHC class I by bovine papillomavirus E5 oncoproteins. Oncogene. 2002, 2: 248-259.
Ashrafi GH, Brown DR, Fife KH, Campo MS: Down-regulation of MHC class I is a property common to papillomavirus E5 proteins. Virus Res. 2006, 120: 208-211.
Faccini AM, Cairney M, Ashrafi GH, Finbow ME, Campo MS, Pitts JD: The bovine papillomavirus type 4 E8 protein binds to ductin and causes loss of gap junctional intercellular communication in primary fibroblasts. J Virol. 1996, 70: 9041-9045.
Andresson T, Sparkowski J, Goldstein DJ, Schlegel R: Vacuolar H(+)-ATPase mutants transform cells and define a binding site for the papillomavirus E5 oncoprotein. J Biol Chem. 1995, 270: 6830-6837.
Ashrafi GH, Pitts JD, Faccini AM , McLean P, O'Brien V, Finbow ME, Campo S: Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events. J Gen Virol. 2000, 81: 689-694.
Roperto S, De Tullio R, Raso C, Stifanese R, Russo V, Gaspari M, Borzacchiello G, Averna M, Paciello O, Cuda G, Roperto F: Calpain3 is espresse in a proteolitically active form in papillomavirus-associated urothelial tumours of the urinary bladder in cattle. PLos One. 2010, 5: e10299-
Kim MK, Kim HS, Kim SH, Oh JM, Han JY, Lim JM, Juhnn YS, Song YS: Human papillomavirus type 16 E5 oncoprotein as a new target for cervical cancer treatment. Biochem Pharmacol. 2010, 80: 1930-1935.
Liu DW, Tsao YP, Hsieh CH, Hsieh JT, Kung JT, Chiang CL, Huang SJ, Chen SL: Induction of CD8 T Cells by Vaccination with Recombinant Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces Tumor Growth. J Virol. 2000, 74: 9083-9089.
Chen YF, Lin CW, Tsao YP, Chen SL: Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumor growth in C57BL/6 mice. J Virol. 2004, 78: 1333-1343.
Lorenzon L, Mazzetta F, Venuti A, Frega A, Torrisi MR, French D: In vivo HPV 16 E5 mRNA: Expression pattern in patients with squamous intra-epithelial lesions of the cervix. J Clin Virol. 2011, 52: 79-83.
Wang W, Sima N, Kong D, Luo A, Gao Q, Liao S, Liao S, Li W, Han L, Wang J, Wang S, Lu Y, Wang D, Xu G, Zhou J, Meng L, Ma D: Selective targeting ofHPV-16 E6/E7 in cervical cancer cells with a potent oncolytic adenovirus and its enhanced effect with radiotherapy in vitro and vivo. Cancer Lett. 2010, 291: 67-75.
Jonson AL, Rogers LM, Ramakrishnan S, Downs LS: Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer. Gynecol Oncol. 2008, 111: 356-364.
Raghava S, Giorda KM, Romano FB, Heuck AP, Hebert DN: The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release. PLoS Pathog. 2011, 7: e1002116-
Maufort JP, Shai A, Pitot HC, Lambert PF: A role for HPV16 E5 in cervical carcinogenesis. Cancer Res. 2010, 70: 2924-2931.
Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H: Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985, 314: 111-114.
Borzacchiello G, Roperto F, Campo MS, Venuti A: 1st International Workshop on Papillomavirus E5 Oncogene-A report. Virology. 2010, 408: 135-137.
MSC thanks Cancer Research UK for its continuous support throughout her career. This work was supported by grant PRIN - Ministero dell'Istruzione, Università e Ricerca Scientifica (MIUR) protocol number 2008LTY389. AV and FP thanks Lega Italiana Lotta Tumori (LILT) for supporting their researches.
The authors declare that they have no competing interests.
All authors have contributed equally to the paper. All authors read and approved the final manuscript.
About this article
Cite this article
Venuti, A., Paolini, F., Nasir, L. et al. Papillomavirus E5: the smallest oncoprotein with many functions. Mol Cancer 10, 140 (2011). https://doi.org/10.1186/1476-4598-10-140
- Cell transformation
- Growth factor receptors
- Immune escape
- E5 oncoprotein
- Animal models