Human Papillomavirus Type 16 E7 Research Articles

Crystal structure of the E2 DNA-binding domain from human papillomavirus type 16: implications for its DNA binding-site selection mechanism

The crystal structure of the E2 DNA-binding domain from the high-risk cervical cancer-associated strain human papillomavirus type 16 (HPV-16) is described here. The papillomavirus E2 proteins regulate transcription from all viral promoters and are required for the initiation of replication in vivo. They belong to a family of viral proteins that form dimeric β-barrels and use surface α-helices for DNA interaction. Although all E2 proteins recognize the same consensus, palindromic DNA sequence, proteins from different viral strains differ in their abilities to discriminate among their specific DNA-binding sites.The structure reported here reveals that while the overall fold of the HPV-16 E2 DNA-binding domain resembles that of its counterpart from the related viral strain bovine papillomavirus type 1, the precise placement of the recognition helices is significantly different. Additionally, the charge distribution on the DNA-binding surfaces of the two proteins varies; HPV-16 E2 has a much less electropositive surface. HPV-16 E2 is thus less able to utilize charge neutralization of the phosphate groups on DNA to induce bending. These results correlate well with previous solution studies that showed decreased affinity between HPV-16 E2 and flexible DNA target sequences, and enhanced affinity towards A-tract-containing, pre-bent sequences. In summary, the crystal structure of the HPV-16 E2 DNA-binding domain shows that the protein presents a stereo-chemically and electrostatically unique surface to DNA, characteristics that can contribute to its mechanism of DNA target discrimination.

A fifteen-amino-acid peptide inhibits human papillomavirus E1-E2 interaction and human papillomavirus DNA replication in vitro.

Mutation of the conserved glutamic acid residue at position 39 of human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its E1 interaction activity and its replication function in transient replication assays but does not affect E2 transcriptional activation. This E39A mutation also disrupts replication activity of HPV-16 E2 in HPV-16 in vitro DNA replication. On this basis, we designed 23- and 15-amino-acid peptides derived from HPV-16 E2 sequences flanking the E39 residue and tested the ability of these peptides to inhibit interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity of these peptides was specific, since analogous peptides in which alanine was substituted for the E39 residue did not inhibit interaction. The 15-amino-acid peptide E2N-WP15 was the smallest peptide tested that effectively inhibited HPV-16 E1-E2 interaction. This peptide also inhibited in vitro replication of HPV-16 DNA. The efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11 and HPV-16 and inhibited in vitro replication with these same combinations of E1 and E2 proteins. These results provide further evidence that E1-E2 interaction is required for papillomavirus DNA replication and constitute the first demonstration that inhibition of this interaction is sufficient to prevent HPV DNA replication in vitro.

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit.

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

Comparison of human papillomavirus type 18 (HPV-18) E6-mediated degradation of p53 in vitro and in vivo reveals significant differences based on p53 structure and cell type but little difference with respect to mutants of HPV-18 E6.

An important characteristic of the E6 proteins derived from oncogenic associated human papillomaviruses (HPVs) is their ability to target the cellular tumour suppressor protein, p53, for ubiquitin mediated degradation. Several studies have attempted to address the important characteristics of both E6 and p53 for this activity in vitro, but the equivalent determinants have not been extensively assessed in vivo. Indeed, recent studies indicate differences between the in vitro and the in vivo degradation assays. We have performed an extensive analysis of the ability of a range of HPV-18 E6 mutants to direct p53 degradation in vivo. In addition, we have also compared the ability of HPV-18 E6 to direct the degradation of different oligomeric forms of p53 both in human and in murine cells. The results of these studies show that mutants of E6 exhibit very similar phenotypes both in vitro and in vivo. In contrast, mutants of p53 show markedly different susceptibilities in vitro and in vivo to E6-induced degradation, and this is further affected by the nature of the cell type in which the assays are performed. Finally, using a cell line temperature sensitive for the E1 ubiquitin-activating enzyme we have been able to show directly that this enzyme is involved in the process of E6-mediated degradation of p53 in vivo.

The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury

Many oncogenes are implicated in the regulation of apoptosis as well as in control of the cell cycle, and several have been shown to protect cells from injury. We tested whether the expression of human papillomavirus type 16 genes E6 and E7 could protect primary astrocytes from injury. Retroviral vectors were used to express E6, E7, or E6E7 in primary murine astrocyte cultures. Astrocytes expressing E6E7 suffered less than half the injury seen in controls after exposure to 400 μM H 2O 2. When we compared astrocytes expressing only E6 or E7, cells expressing E7 alone were protected to a greater extent and from more severe injury than those expressing E6. E6E7, E6 and E7 all provided protection from 30 h glucose deprivation, but again E7 provided the best protection, reducing injury to less than a third of that seen in controls. Expression of E7 alone decreases vulnerability to both hydrogen peroxide and glucose deprivation injury while E6 alone primarily decreases glucose deprivation injury. These results demonstrate that expression of human papillomavirus oncogenes can reduce the vulnerability of astrocytes to oxidative stress and nutrient deprivation.

Transcription factor YY1 represses cell-free replication from human papillomavirus origins.

We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (ori)-containing DNA by using purified HPV-18 E1 and E2 gene products expressed as fusion proteins in Escherichia coli. The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18 ori. We show that exogenously added YY1 fusion protein inhibited HPV-18 ori replication. Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells. However, inhibition did not appear to be mediated by binding to its cognate site in the ori as YY1 also inhibited the replication of the HPV-11 ori, which does not have a known or suspected YY1 binding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration. These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.

Construction of artificial epithelial tissues prepared from human normal fibroblasts and C9 cervical epithelial cancer cells carrying human papillomavirus type 18 genes

One cervical cancer cell line, C9, carrying human papillomavirus type 18 (HPV18) genes that is one of the major etiologic oncoviruses for cervical cancer was characterized. This cell line was further characterized for its capacity related to the epithelial cell proliferation, stratification and differentiation in reconstituted artificial epithelial tissue. Thein vitro construction of three dimensional artificial cervical epithelial tissue has been engineered using C9 epithelial cancer cells, human foreskin fibroblasts and a matrix made of type I collagen by organotypic culture of epithelial cells. The morphology of paraffin embedded artificial tissue was examined by histochemical staining. The artificial epithelial tissues were well developed having multilayer. However, the tissue morphology was similar to the cervical tissue having displasia induced by HPV infection. The characteristics of the artificial tissues were examined by determining the expression of specific marker proteins. In the C9 derived artificial tissues, the expression of EGF receptor, an epithelial proliferation marker proteins for stratum basale was observed up to the stratum spinosum. Another epithelial proliferation marker for stratum spinosum, cytokeratins 5/6/18, were observed well over the stratum spinosum. For the differentiation markers, the expression of involucrin and filaggrin were observed while the terminal differentiation marker, cytokeratins 10/13 were not detected at all. Therefore the reconstituted artificial epithelial tissues expressed the same types of differentiation marker proteins that are expressed in normal human cervical epithelial tissues but lacked the final differentiation capacity representing characteristics of C9 cell line as a cancer tissue derived cell line. Expression of HPV18 E6 oncoprotein was also observed in this artificial cervical epithelial tissue though the intensity of the staining was weak. Thus this artificial cervical epithelial tissue though the intensity of the staining was weak. Thus this artificial epithelial tissue could be used as a useful model system to examine the relationship between HPV-induced cervical oncogenesis and epithelial cell differentiation.

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.

The Early HPV16 Proteins Can Regulate mRNA Levels of Cell Cycle Genes in Human Cervical Carcinoma Cells by p53-Independent Mechanisms

Cervical carcinoma-associated human papillomavirus type 16 (HPV16) encodes E6 and E7 oncoproteins which inactivate p53 and Rb, respectively, but these interactions are not sufficient to account for the oncogenic potential of the virus. Several viral promoters were shown to be regulated by E6 and E7. To identify genes as cellular targets of the HPV16 early proteins, we transfected a new HPV-negative and p53-mutated cervical carcinoma-derived cell line with either the HPV16 full-length genome or the HPV16 E6 gene. HPV16 clones but not 16E6 clones showed a decreased doubling time that was not related to the viral DNA and mRNA patterns. In exponentially growing cells as well as in cells synchronized by serum starvation, expression of the E6 gene was associated with upregulation of the c-fosand c-junproto-oncogenes and with downregulation of the c-Ha-rasgene. Furthermore, a viral gene other than E6 may be involved in downregulation of p53 because a reduced mRNA level at the G1/S transition was observed only in HPV16-cells. The present study on natural host cells indicates p53-independent transcriptional modulations of cell cycle regulatory genes related to HPV16 E6 and E7 expression.

The Human Papillomavirus Type 18 (HPV18) Replication Protein E1 Is a Transcriptional Activator When Interacting with HPV18 E2

The human papillomavirus type 18 E1 and E2 proteins are both required for the initiation of viral DNA replication. Whereas E2 is the major viral transcription regulator, E1 is the replication initiator protein. They interact with each other and with the origin sequences to initiate viral DNA replication. We show that the HPV18 E1 and E2 proteins, when bound to an origin sequence cloned upstream of a heterologous promoter, synergistically activate transcription. This synergy required binding of E2 to at least two binding sites, but was partially independent of E1 binding to the origin of replication. Transcriptional activation was observed even in the absence of replication of the target DNA. Only homologous E1 and E2 proteins binding to homologous origin sequences from BPV1 or HPV18 viruses could synergistically activate transcription. We show that the HPV18 E1 protein can activate transcription when targeted to the DNA by fusion of the complete polypeptide with the BPV1 E2 C-terminus dimerization/DNA binding domain, implying that HPV18 E1 is an intrinsic transcriptional activator, though less potent than E2. The interaction between E1 and E2 may form a transcriptionally active complex during initiation of viral DNA replication.

Disruption of the G1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin E.

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

Disruption of the human papillomavirus type 16 E2 gene protects cervical carcinoma cells from E2F-induced apoptosis.

Human papillomavirus type 16 (HPV-16) is a DNA tumour virus that has been implicated in the development of cervical cancer. In non-transformed HPV-infected cells, the HPV E2 protein regulates transcription of the viral E6 and E7 oncogenes. Malignant transformation is usually accompanied by disruption of the E2 gene and consequent deregulated expression of E6 and E7. Here we show that re-introduction of the HPV-16 E2 protein into an HPV-16-transformed cervical carcinoma cell line results in a decrease in growth rate and, in the absence of serum growth factors, cell death via apoptosis. E2 expression increases E6/E7 mRNA levels. This brings about an increase in E7 protein levels, which in turn leads to an increase in free E2F, a condition that has previously been shown to induce apoptotic cell death. Despite the increase in E6 mRNA there is no detectable E6 protein in these cells and E2 expression does not reduce the activity of a p53-responsive promoter. Our data suggest that disruption of the E2 gene produces HPV-transformed cells that are less liable to undergo apoptosis and, therefore, more likely to form cervical tumours.

Cytokeratin patterns of expression in human epithelial cell lines correlate with transcriptional activity of the human papillomavirus type 16 upstream regulatory region.

A comparison of the CAT reporter activity of a plasmid which contains the 232 bp epithelial specific enhancer alone with that of plasmids which contain additional sequences from the human papillomavirus type 16 (HPV-16) upstream regulatory region (URR) revealed two markedly different patterns, in an analysis of six human epithelial cell lines. In HeLa, C33A and SiHa, the CAT reporter activities of all the constructs were comparable. In contrast, in CaSki, HK2bE6-E7 and HaCaT we detected very low levels of CAT reporter activity using the constructs with the additional HPV-16 URR sequences. The ability of HPV-16 E2 to transactivate a construct with 2 E2 binding sites also differed markedly and showed the same pattern. Cytokeratin staining revealed a correlation between cytokeratin 10 and 14 expression and the transcriptional differences observed. We also found alterations in the activity of one of the constructs on altering the growth conditions of the HaCaT cell line.

Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia.

Infection with human papillomavirus (HPV) type 16 (HPV16) is a major cause of high-grade cervical intraepithelial neoplasia (CIN). Experiments were planned to evaluate the role of cell-mediated immunity (e.g., lymphocyte proliferation) against HPV in the natural history of HPV-associated neoplasia and to identify antigenic sequences of the HPV16 proteins E6 and E7 against which an immune response may confer protection. Forty-nine women with abnormal cervical cytology and biopsy-confirmed CIN were followed through one or more clinic visits. Lymphoproliferative responses of peripheral blood mononuclear cells to HPV16 E6 and E7 peptides were assessed in long-term (3-week) cultures. HPV DNA was detected in cervicovaginal lavage by means of polymerase chain reaction and Southern blotting. Disease status was determined by cervical cytologic examination and colposcopy. Reported P values are two-sided. Subjects with positive lymphoproliferative responses to E6 and/or E7 peptides were more likely to be HPV negative at the same clinic visit than were nonresponders (P = .039). Subjects who were negative for HPV and those with a low viral load were more likely to be responders than were those with a high viral load (P for trend = .037). Responses to N-terminal E6 peptide 369 were associated with absence of HPV infection at the same clinic visit (P = .015). Subjects with positive responses to E6 or E7 peptides at one clinic visit were 4.4 times more likely to be HPV negative at the next visit than were nonresponders (P = .142). Responses to E6 peptide 369 and/or E7 C-terminal peptide 109 were associated with an absence of HPV infection (P = .02 for both) and an absence of CIN (P = .04 and .02, respectively) at the next visit. Lymphoproliferative responses to specific HPV16 E6 and E7 peptides appear to be associated with the clearance of HPV infection and the regression of CIN.

Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.

Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins.

Random mutagenesis of human papillomavirus type 16 (HPV16) E1 was used to generate E1 missense mutants defective for interaction with either hUBC9 or 16E1-BP, two cDNAs encoding proteins that have been identified by their ability to interact with HPV16 E1 in two-hybrid assays. hUBC9, the human counterpart of Saccharomyces cerevisiae UBC9, is a ubiquitin-conjugating enzyme known to be involved in cell cycle progression. 16E1-BP encodes a protein of no known function but does contain an ATPase signature motif. Eight hUBC9 or 16E1-BP interaction-defective HPV16 E1 missense mutants were identified and characterized for origin-dependent transient DNA replication, ATPase activity, and various protein-protein interaction phenotypes. Six of these mutant E1 proteins were significantly impaired for replication. Among these, two classes of replication-defective HPV16 E1 missense mutants were observed. One class, represented by the S330R replication-defective mutant (containing an S-to-R change at position 330), remained competent for all protein-protein interactions tested, with the exception of hUBC9 association. Furthermore, this mutant, unlike the other replication-defective HPV16 E1 missense mutants, had a strong dominant negative replication phenotype in transient-replication assays. The other class, represented by five of the missense mutants, was defective for multiple protein-protein interactions, usually including, but not limited to, the interaction defect for which each mutant was originally selected. In many cases, a single missense mutation in one region of HPV16 E1 had pleiotropic effects, even upon activities thought to be associated with other domains of HPV16 E1. This suggests that E1 proteins are not modular but may instead be composed of multiple structurally and/or functionally interdependent domains.

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.

Presentation of the HPV16E7 Protein by Skin Grafts Is Insufficient to Allow Graft Rejection in an E7-Primed Animal

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the αA crystallin promoter (FVBαAcryHPV16E6E7). We have transferred skin from FVBαAcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB × C57BL/6J mice recipients of FVBαAcryHPV16E6E7 × C57BL/6J skin generated humoral and DTH responses to E7in vivoand E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

Two distinct human uterine cervical epithelial cell lines established after transfection with human papillomavirus 16 DNA.

We have established two distinct human cervical cell lines, NCC16 and NCE16, after traiisfecting human papillomavirus type 16 (HPV16) DNA into normal human ecto‐cervical and endo‐cervical epithelial cells, respectively. Both lines expressed HPV16 E6 and E7 as detected hy reverse transcriptase‐polymerase chain reaction and northern Wot hybridization. These cells have been passaged for over 100 population doublings and express strong telomerase activity. Neither cell line was tumorigenic in athymic nu/nu mice. However, both NCC16 and NCE16 developed abnormally stratified architectures following implantation with a silicon membrane sheet in the back of athymic nude mice. The former cells were pathohistologically similar to carcinoma, while the latter produced Alcian‐blue positive cells, suggesting the occurrence of metaplastic changes. These distinct cell lines offer a useful model system for the study of cervical carcinogenesis and of its regulatory mechanism after HPV infection in different regions of the uterine cervix.