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.     

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.     

Human papillomavirus type 16 E7 oncoprotein binds and inactivates growth-inhibitory insulin-like growth factor binding protein 3

The E7 protein encoded by human papillomavirus type 16 is one of the few viral genes that can immortalize primary human cells and thereby override cellular senescence. While it is generally assumed that this property of E7 depends on its interaction with regulators of the cell cycle, we show here that E7 targets insulin-like growth factor binding protein 3 (IGFBP-3), the product of a p53-inducible gene that is overexpressed in senescent cells. IGFBP-3 can suppress cell proliferation and induce apoptosis; we show here that IGFBP-3-mediated apoptosis is inhibited by E7, which binds to IGFBP-3 and triggers its proteolytic cleavage. Two transformation-deficient mutants of E7 failed to inactivate IGFBP-3, suggesting that inactivation of IGFBP-3 may contribute to cell transformation.     

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.     

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.     

Chaperone holdase activity of human papillomavirus E7 oncoprotein

E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.     

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.     

Computer-assisted analysis of molecular mimicry between human papillomavirus 16 E7 oncoprotein and human protein sequences

The immunology of human papillomavirus (HPV) infections has peculiar characteristics. The long latency for cervical cancer development after primary viral infection suggests mechanisms that may aid the virus in avoiding the host immunosurveillance and establishing persistent infections. In order to understand whether molecular mimicry phenomena might explain the ability of HPV to avoid a protective immune response by the host cell, sequence similarity between HPV16 E7 oncoprotein and human self-proteins was examined by computer-assisted analysis. Data were obtained showing that the HPV16 E7 protein has high and widespread similarity to several human proteins involved in a number of critical regulatory processes. In addition, multiple identical and different E7 peptide motifs are present in the same human protein. Thus, sharing of common motifs between viral oncoproteins and molecules of normal cells may be one cause underlying the scarce immunogenicity of HPV infections. The hypothesis is advanced that synthetic peptides harbouring viral motifs not and/or scarcely represented in the host's cellular proteins may represent a valuable immunotherapeutic approach for cervical cancer treatment.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

The human papillomavirus type 16 E5 oncoprotein translocates calpactin I to the perinuclear region

The human papillomavirus type 16 (HPV-16) E5 oncoprotein is embedded in membranes of the endoplasmic reticulum and nuclear envelope with its C terminus exposed to the cytoplasm. Among other activities, E5 cooperates with the HPV E6 oncoprotein to induce koilocytosis in human cervical cells and keratinocytes in vitro. The effect of E5 on infected cells may rely on its interactions with various cellular proteins. In this study we identify calpactin I, a heterotetrameric, Ca(2+)- and phospholipid-binding protein complex that regulates membrane fusion, as a new cellular target for E5. Both the annexin A2 and p11 subunits of calpactin I coimmunoprecipitate with E5 in COS cells and in human epithelial cell lines, and an intact E5 C terminus is required for binding. Moreover, E5-expressing cells exhibit a perinuclear redistribution of annexin A2 and p11 and show increased fusion of perinuclear membrane vesicles. The C terminus of E5 is required for both the perinuclear redistribution of calpactin I and increased formation of perinuclear vacuoles. These results support the hypothesis that the E5-induced relocalization of calpactin I to the perinuclear region promotes perinuclear membrane fusion, which may underlie the development of koilocytotic vacuoles.     

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.     

Bovine papillomavirus E7 oncoprotein inhibits anoikis

The bovine papillomavirus type 1 (BPV-1) E7 oncoprotein is required for the full transformation activity of the virus. Although BPV-1 E7 by itself is not sufficient to induce cellular transformation, it enhances the abilities of the other BPV-1 oncogenes to induce anchorage independence. We have been exploring the mechanisms by which E7 might affect the transformation efficiency of other viral oncoproteins and in particular whether it might protect cells from apoptosis. We report here that BPV-1 E6 and E7 can each independently inhibit anoikis, a type of apoptosis that is induced upon cell detachment. Using site-directed mutagenesis, we determined regions of the E7 protein that were essential for its antiapoptotic activity. The ability of E7 to inhibit anoikis did partially correlate with an ability to enhance anchorage independence of BPV-1 E6-transformed cells. In addition, the antiapoptotic activity of E7 also only partially correlated with its ability to bind p600, a cellular protein that has previously been reported to play a role in anoikis. We conclude that the contribution of E7 to BPV-induced cellular transformation may involve its ability to inhibit anoikis but that additional functional activities must also be involved.     

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 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.