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.     

Replication of human papillomavirus (HPV) DNAs supported by the HPV type 18 E1 and E2 proteins.

Transient replication of human papillomavirus (HPV) type 18 DNA was shown to require the viral E1 and E2 proteins. A 108-bp sequence within the long control region (nucleotides 12 to 119) was sufficient to function as the origin, but maximal replication required a region of 177 bp from positions 7800 to 7857 and 1 to 119 of HPV-18. The E1 and E2 proteins of HPV-18 also supported transient replication of plasmids containing the origins of HPV-1a and bovine papillomavirus type 1 to low levels. Interestingly, the level of replication observed with the HPV-6b origin was higher than that obtained with the homologous HPV-18 origin.     

Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.     

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.     

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.     

A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein

Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or glutathione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties ('soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.     

Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.     

The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins

We have recently identified E6TP1 (E6-targeted protein 1) as a novel high-risk human papillomavirus type 16 (HPV16) E6-binding protein. Importantly, mutational analysis of E6 revealed a strong correlation between the transforming activity and its abilities to bind and target E6TP1 for ubiquitin-mediated degradation. As a region within E6TP1 has high homology with GAP domains of known and putative Rap GTPase-activating proteins (GAPs), these results raised the possibility that HPV E6 may alter the Rap small-G-protein signaling pathway. Using two different approaches, we now demonstrate that human E6TP1 exhibits GAP activity for Rap1 and Rap2, confirming recent findings that a closely related rat homologue exhibits Rap-specific GAP activity. Using mutational analysis, we localize the GAP activity to residues 240 to 945 of E6TP1. Significantly, we demonstrate that coexpression of HPV16 E6, by promoting the degradation of E6TP1, enhances the GTP loading of Rap. These results support a role of Rap small-G-protein pathway in E6-mediated oncogenesis.     

E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.     

Human papillomavirus (HPV) oncoprotein E6 facilitates Calcineurin-Nuclear factor for activated T cells 2 (NFAT2) signaling to promote cellular proliferation in cervical cell carcinoma

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.     

Domain substructure of HPV E6 oncoprotein: biophysical characterization of the E6 C-terminal DNA-binding domain

E6 is a viral oncoprotein implicated in cervical cancers, produced by high-risk human papillomaviruses (HPVs). Structural data concerning this protein are scarce due to the difficulty of producing recombinant E6. Recently, we described the expression and purification of a stable, folded, and biologically active HPV16 E6 mutant called E6 6C/6S. Here, we analyzed the domain substructure of this mutated E6. Nonspecific proteolysis of full-length E6 6C/6S (158 residues) yielded N-terminal and C-terminal fragments encompassing residues 7-83 and 87-158, respectively. The C-terminal fragment of residues 87-158 was cloned, overexpressed, and purified at concentrations as high as 1 mM. The purified domain retains the selective four-way DNA junction recognition activity of the full-length E6 protein. Using UV absorption, UV fluorescence, circular dichroism, and nuclear magnetic resonance, we show that the peptide is primarily monomeric and folded with equal proportions of alpha-helix and beta-sheet secondary structure.     

Human papillomavirus 16 E6 oncoprotein interferences with insulin signaling pathway by binding to tuberin

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either TSC1 or TSC2 tumor suppressor gene. TSC1 and TSC2 products, Harmatin and Tuberin, form the functional complex to serve as the negative regulator for insulin-induced phosphorylation of S6 kinase and elF4E-binding protein 1. High-risk human papillomavirus (HPV) infection is the necessary cause for cervical cancer. E6 oncoprotein encoded by HPV plays a pivotal role in carcinogenesis by interference with the host intracellular protein functions. In this study, we show that HPV16 E6 interacts with tumor suppressor gene TSC2 product, Tuberin, and results in the phosphorylation of S6 kinase and S6 even in the absence of insulin. The overexpression of Tuberin overcomes the effect of E6 on S6 kinase phosphorylation. Binding with HPV16 E6 causes the proteasome-mediated degradation of Tuberin. A DILG motif and an ELVG motif located in the carboxyl-terminal of Tuberin are required for E6 binding. In addition, the Tuberin interaction region in E6 has been mapped in the amino-terminal portion of HPV16 E6, which is different from the binding domain with p53. These results provide a possible link between E6-induced oncogenesis and the insulin-stimulated cell proliferation signaling pathway.