Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein

The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.Human papillomaviruses (HPVs) are small, nonenveloped, double-stranded DNA viruses (25) that are the causative agents of benign and malignant tumors in humans (43). Most cancers of the cervix, vagina, and anus are caused by HPVs, as are a fraction of oropharyngeal cancers (29, 44). HPV type 16 (HPV-16) is the type most frequently found in anogenital cancers (15, 29), including cervical cancer, the most common cancer of women worldwide (44).Some of the biological activities of the HPV-16 E5 protein (16E5) include the augmentation of epidermal growth factor (EGF) signaling pathways (8), stimulation of anchorage-independent growth (38), alkalinization of endosomal pH (11), and alteration of membrane lipid composition (39). 16E5 also exhibits weak transforming activity in vitro (12), induces epithelial tumors in transgenic mice (13), and plays an important role in koilocytosis (20). There are multiple documented intracellular binding targets for 16E5 such as the 16-kDa subunit of the vacuolar H⁺-ATPase (7, 36), the heavy chain of HLA type I (1), EGF receptor family member ErbB4 (6), calnexin (16), the zinc transporter ZnT-1 (21), the EVER1 and EVER2 transmembrane channel-like proteins that modulate zinc homeostasis (21, 31), the nuclear import receptor family member karyopherin β3 (KNβ3) (19), and BAP31, which was previously reported to contribute to B-cell receptor activation (35).16E5 is a small, hydrophobic protein that localizes to intracellular membranes. When overexpressed in COS cells, it is present in the endoplasmic reticulum (ER) and, to a lesser extent, in the Golgi apparatus (7). At a lower level of expression in human foreskin keratinocytes and human ectocervical cells (HECs), 16E5 is present predominantly in the ER (10, 39). 16E5 contains three hydrophobic regions (14, 16, 22, 30, 41), and it was reported previously that the first hydrophobic region determines various biological properties of the protein (16, 22). It was also shown previously that the 16E5 C terminus plays a role in binding to karyopherin β3 (19) and in the formation of koilocytes (20). While theoretical predictions have been made for the topology of E5 in membranes (16), no experimental data exist. However, a recent study suggested that some highly expressed 16E5 localizes to the plasma membrane, with its C terminus exposed externally (18).The aim of the present study was to establish the orientation of 16E5 in the ER membrane. By using immunofluorescence microscopy coupled with differential membrane permeabilization (24, 34), we demonstrate the membrane orientation of an N- and C-terminally tagged, biologically active 16E5 protein. Our results indicate that the N terminus is intralumenal and that the C terminus is cytoplasmic, consistent with a model of E5 being a three-pass transmembrane protein and with current data on the interaction of its C terminus with cytoplasmic proteins.     

The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A₁ (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.High-risk human papillomaviruses (HPVs) are the causative agent of cervical cancer (63) and HPV type 16 (HPV-16) is associated with a majority of cervical malignancies worldwide (13). HPV-16 encodes three oncoproteins: E5, E6, and E7. While the contributions of E6 and E7 to cellular immortalization and transformation have been characterized in detail (20), the role of HPV-16 E5 (16E5) is poorly understood (53). Nevertheless, a number of studies suggest that 16E5 does contribute to the development of cervical cancer. Most high-risk HPV types encode an E5 protein (48), and targeted expression of the three HPV-16 oncogenes in basal epithelial cells of transgenic mice (4) leads to a higher incidence of cervical cancer than does the expression of E6 and E7 alone (44). In addition, targeted epithelial expression of 16E5 (without E6 and E7) in transgenic mice induces skin tumors (21). It may be noteworthy that unlike high-risk HPV-18, which integrates into the host DNA and potentially disrupts E5 gene expression (20, 64), the HPV-16 genome often persists in episomal form in malignant lesions (12, 16, 24, 36, 42).Biological activities of 16E5 that may facilitate carcinogenesis include evading host immune detection by interfering with the transport of antigen-presenting major histocompatibility complex (MHC) class I molecules to the cell surface (6), promoting anchorage-independent growth (33, 41, 52) and disrupting gap junctions responsible for cell-cell communication (37, 58). The 16E5 phenotype most frequently linked to the development of cancer is enhanced ligand-dependent activation of the epidermal growth factor receptor (EGFR) (15, 41, 46, 52). 16E5 stimulates EGF-dependent cell proliferation in vitro (7, 33, 40, 41, 52, 60) and in vivo (21), which might expand the population of basal or stemlike keratinocytes and thereby increase the probability that some of these cells would undergo malignant transformation. A number of studies indicate that 16E5 may enhance ligand-dependent EGFR activation by interfering with the acidification of early endosomes containing EGF bound to activated EGFRs (17, 51, 57). It has been hypothesized that 16E5 inhibits the H⁺ V-ATPase responsible for maintaining an acidic luminal pH in late endosomes and lysosomes (28) by associating with the V-ATPase 16-kDa “c” subunit (16K) (1, 5, 14, 22, 46) and disrupting assembly of the V-ATPase integral (V_o) and peripheral (V_i) subcomplexes (10). In contrast, Thomsen et al. (57) reported that 16E5 inhibits early endosome trafficking in fibroblasts by completely depolymerizing actin microfilaments.Due to the unavailability of antibodies that recognize native 16E5 and 16K, direct association of 16E5 with 16K has only been observed by overexpressing epitope-tagged forms of both proteins in vitro (5, 46) or in vivo (1, 14, 22). It is uncertain, therefore, whether these associations occur when the proteins are expressed at “physiological” levels. In yeast, both wild-type 16E5 (10) and several 16E5 mutants that associate with 16K in COS cells (1) inhibit vacuolar acidification, although another study in yeast concludes the opposite (5). 16K is a component of the V-ATPase V_o subcomplex, which is assembled in the endoplasmic reticulum (ER) (28), and 16E5 localizes to the ER and nuclear envelope in epithelial cells (32, 54). Thus, the export of V_o from the ER could potentially be inhibited by a significant level of 16K binding to 16E5, although the differential alkalinization of endosomes rather than the Golgi apparatus (17) would require specificity for those proton pumps directed to those sites.In the present study, we generated an antibody against native 16K and used it to determine whether 16K/16E5 complexes formed in primary keratinocytes. We also synthesized a new pH-sensitive fluorescent EGF conjugate to evaluate whether there was a correlation between E5-induced EGFR activation, trafficking and endosome alkalinization. Finally, we simultaneously monitored EGFR endocytic trafficking (using pH-insensitive fluorescent EGF), endosome fusion (using fluorescent EGF and dextran), and the status of cellular filaments and microtubules to evaluate whether E5 might disrupt some of these structures that mediate vesicle transport.     

The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.     

Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.     

The HPV16 E6 Oncoprotein Causes Prolonged Receptor Protein Tyrosine Kinase Signaling and Enhances Internalization of Phosphorylated Receptor Species

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

E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal growth factor receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human keratinocytes. Phosphatidylinositol 3-kinase (PI3K) and ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival factor. To test this hypothesis, the apoptotic response of UV B-irradiated primary keratinocytes infected with either control retrovirus, LXSN, or HPV16 2E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/2 MAPK pathways, respectively, suggesting that the PI3K and ERK1/2 MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/2 MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/2 activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were growth arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/2 MAPK signal pathways.     

Evidence of Diversifying Selection in Human Papillomavirus Type 16 E6 But Not E7 Oncogenes

Human papillomavirus type 16 is a common sexually transmitted pathogen capable of giving rise to cervical intraepithelial neoplasia and invasive carcinoma through the expression and activity of two adjacent oncogenes: E6 and E7. Naturally occurring amino acid variation is commonly observed in the E6 protein but to a much lesser extent in E7. In order to investigate the evolutionary mechanisms involved in the generation and maintenance of this variation, we examine 42 distinct E6-E7 haplotypes using codon-based genealogical techniques. These techniques involve estimation of the ratio of nonsynonymous to synonymous substitutions (dn/ds) and allow testing for directional (positive) natural selection. Positive selection was detected for four codon sites within the E6 oncogene but not in any E7 codons. The amino acid compositions and locations of selected sites are described. Possible sources of natural selection including antiviral immune pressure and polymorphism of host cellular proteins are discussed.     

人乳头瘤病毒58型E6E7融合蛋白的原核表达、纯化及鉴定

目的:构建pET-42a(+)-HPV58E6E7原核表达质粒,诱导表达人乳头瘤病毒(HPV)58型E6E7融合蛋白。方法:采用PCR方法扩增出HPV58 E6E7融合基因的全长序列,利用DNA重组技术将其定向插入原核表达载体pET-42a(+)中,构建pET-42a(+)-HPV58E6E7原核表达质粒,用限制性内切酶酶切和核酸序列检测对重组质粒进行鉴定;将其转入宿主菌大肠杆菌BL21进行诱导以表达HPV58E6E7融合蛋白,并用谷胱甘肽琼脂糖树脂纯化回收HPV58E6E7融合蛋白,用SDS-PAGE及Western印迹鉴定表达蛋白的相对分子质量及抗原性。结果:PCR、限制性内切酶酶切和核酸序列检测证实重组质粒中插入的目的基因大小、方向正确;HPV58E6E7融合蛋白得到高效原核表达及纯化,表达蛋白的分子大小正确,抗原性良好。结论:pET-42a(+)-HPV58E6E7原核表达质粒构建成功,HPV58E6E7融合蛋白得到高效表达及有效纯化,为检测HPV58型治疗性疫苗的免疫效果提供了抗原。     

云南省人乳头状瘤病毒16型E6/E7基因的变异分析

研究人乳头状瘤病毒16型E6和E7基因在云南省的变异情况。采集获得2 000例妇科门诊样品,提取DNA,以MY09/MY11为外引物,GP5+/GP6+为内引物,采用nest-PCR法对样品HPV-DNA高变区L1区的相应基因进行扩增、测序和分型,筛选得到20例HPV-16型病毒DNA,对其进行E6和E7基因特异性扩增,测序。结果显示20例HPV-16型E6基因中有10例在178位核苷酸发生碱基突变,突变频率为50%,E7基因中有10例在647位核苷酸发生碱基突变,突变频率为50%。进化树分析结果表明在云南省流行的HPV-16型中主要为亚洲变异型,没有发现非洲1型,非洲2型。     

The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.     

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.