The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.     

Modelling of the human papillomavirus type 16 E5 protein

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.     

Mutational analysis of human papillomavirus type 11 E5a oncoprotein.

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes.

Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.     

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.     

Human papillomavirus type 16 E7 oncoprotein associates with the centrosomal component gamma-tubulin

Expression of a high-risk human papillomavirus (HPV) E7 oncoprotein is sufficient to induce aberrant centrosome duplication in primary human cells. The resulting centrosome-associated mitotic abnormalities have been linked to the development of aneuploidy. HPV type 16 (HPV16) E7 induces supernumerary centrosomes through a mechanism that is at least in part independent of the inactivation of the retinoblastoma tumor suppressor pRb and is dependent on cyclin-dependent kinase 2 activity. Here, we show that HPV16 E7 can concentrate around mitotic spindle poles and that a small pool of HPV16 E7 is associated with centrosome fractions isolated by sucrose density gradient centrifugation. The targeting of HPV16 E7 to the centrosome, however, was not sufficient for centrosome overduplication. Nonetheless, we found that HPV16 E7 can associate with the centrosomal regulator γ-tubulin and that the recruitment of γ-tubulin to the centrosome is altered in HPV16 E7-expressing cells. Since the association of HPV16 E7 with γ-tubulin is independent of pRb, p107, and p130, our results suggest that the association with γ-tubulin contributes to the pRb/p107/p130-independent ability of HPV16 E7 to subvert centrosome homeostasis.     

Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells

Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.     

Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins.

The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.     

Golgi alkalinization by the papillomavirus E5 oncoprotein

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.     

Localisation of human papillomavirus 16 E7 oncoprotein changes with cell confluence

E7 is one of the best studied proteins of human papillomavirus type 16, largely because of its oncogenic potential linked to cervical cancer. Yet the sub-cellular location of E7 remains confounding, even though it has been shown to be able to shuttle between the nucleus and the cytoplasm. Here we show with immunocytochemistry that E7 proteins are located in the nucleus and cytoplasm in sub-confluent cells, but becomes cytoplasmic in confluent cells. The change in E7's location is independent of time in culture, cell division, cell cycle phase or cellular differentiation. Levels of E7 are also increased in confluent cells as determined by Western blotting. Our investigations have also uncovered how different analytical techniques influence the observation of where E7 is localised, highlighting the importance of technical choice in such analysis. Understanding the localisation of E7 will help us to better comprehend the function of E7 on its target proteins.     

High-risk human papillomavirus E5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors

High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded "viroporin" family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established de novo molecular modeling and design methods permitted the development of the first specific small-molecule E5 inhibitor, capable of both abrogating channel activity in vitro and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.     

Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.     

Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16.

Transfection of the E6 and E7 genes of the high-risk human papillomaviruses (HPVs) into primary genital keratinocytes generates colonies of proliferating cells which are resistant to calcium- and serum-induced terminal differentiation. To genetically map the HPV gene(s) responsible for this cellular phenotype, we cloned cDNAs for full-length E6, full-length E7, four truncated E6 splice variants (E6I to E6IV), and a series of E6 C-terminal truncation mutants into a simian virus 40 expression vector. The E6 proteins were tagged with the AU1 epitope at the C terminus to verify their expression in COS cells by immunoprecipitation and immunofluorescence. Transfection of the full-length E6 protein, either wild type or epitope tagged, induced calcium- and serum-resistant keratinocyte colonies, but the truncated E6 variants and full-length E7 protein did not. E6-induced colonies, while altered in response to serum and calcium, could not be established into cell lines without the combined presence of the E7 protein, further exemplifying the independent roles of E6 and E7 in cell differentiation and cell proliferation. The E6 C-terminal deletion mutants defined two distinct functional domains between amino acids 120 and 151. Amino acids 120 to 151 contained an apparent bipartite nuclear localization signal, whereas amino acids 132 to 141 were required for the induction of resistance to calcium- and serum-induced differentiation and for immortalization of human keratinocytes in conjunction with E7.