Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes.

The E5/E8 hydrophobic protein of BPV-4 is, at only 42 residues, the smallest transforming protein identified to date. Transformation of NIH-3T3 cells by E5/E8 correlates with up-regulation of both cyclin A-associated kinase activity and, unusually, p27(Kip1) (p27) but does not rely on changes in cyclin E or cyclin E-CDK2 activity. Here we have examined how p27 is prevented from functioning efficiently as a CDK2 inhibitor, and we investigated the mechanisms used to achieve elevated p27 expression in E5/E8 cells. Our results show that normal subcellular targeting of p27 is not subverted in E5/E8 cells, and p27 retains its ability to inhibit both cyclin E-CDK2 and cyclin A-CDK activities upon release from heat-labile complexes. E5/E8 cells also have elevated levels of cyclins D1 and D3, and high levels of nuclear p27 are tolerated because the inhibitor is sequestered within an elevated pool of cyclin D1-CDK4 complexes, a significant portion of which retain kinase activity. In agreement with this, pRB is constitutively hyperphosphorylated in E5/E8 cells in vivo. The increased steady-state level of p27 is achieved largely through an increased rate of protein synthesis and does not rely on changes in p27 mRNA levels or protein half-life. This is the first report of enhanced p27 synthesis as the main mechanism for increasing protein levels in continuously cycling cells. Our results are consistent with a model in which E5/E8 promotes a coordinated elevation of cyclin D1-CDK4 and p27, as well as cyclin A-associated kinase activity, which act in concert to allow continued proliferation in the absence of mitogens.

A mutational analysis of the transforming functions of the E8 protein of bovine papillomavirus type 4.

The E8 protein of BPV-4 contributes to transformation of primary bovine cells (PalFs) by inducing anchorage-independent growth and by down-regulating gap junction intercellular communication, likely due to its binding to 16K ductin. We show here that, in addition, E8 confers on PalF cells the ability to grow in low serum and to escape from contact inhibition (focus formation). E8 also transactivates an exogenous human cyclin A gene promoter, suggesting that overexpression of cyclin A is responsible for the transformed phenotype. Mutant forms of E8 were generated to establish whether the transforming functions of the protein could be segregated. Mutations were introduced both in the hydrophobic domain and in the hydrophilic C-terminal "tail", and chimeras with BPV-1 E5 were constructed. Cells expressing either wild-type E8 or mutant forms were analyzed for their ability to grow in low serum and in suspension and to form foci. Wild-type E8 and its mutants were also analyzed for their ability to transactivate the cyclin A promoter. We show here that the transforming functions of E8 can be segregated and that both the hydrophilic C-terminal tail and the residue at position 17 in the hydrophobic domain are crucial for E8 functions and for the transactivation of the cyclin A promoter. These results support the hypothesis that the different aspects of cellular transformation brought about by E8 might be due to interaction with different cellular targets. They suggest that E8 might function differently from BPV-1 E5 and demonstrate that the separate domains of E5 and E8 are not functionally interchangeable.

The bovine papillomavirus type 4 long control region contains an epithelial specific enhancer.

Bovine papillomavirus type 4 (BPV-4) is a mucosal epitheliotropic virus that is a causative agent in alimentary carcinoma of cattle. The long control region (LCR) of this virus controls expression of the transforming proteins, E8 and E7. Deletion mutants of the LCR were prepared and assayed for their ability to activate transcription from the LCR promoter in primary bovine palate keratinocytes (the natural target cell for BPV-4) and fibroblasts. The LCR was at least an order of magnitude more active in keratinocytes than in fibroblasts. An epithelial specific enhancer was identified that activated transcription from the SV40 promoter to levels identical to the full-length LCR. One of the active sites in the enhancer is 100% conserved in the LCR of human papillomavirus type 16. The results demonstrate that the BPV-4 LCR has an epithelial specific enhancer, which offers the opportunity to study epithelial specific transcriptional regulation of papillomavirus promoters.

Epithelial specific transcriptional regulation of the bovine papillomavirus 4 promoter by E2.

Bovine papillomavirus 4 (BPV-4) is a mucosal epitheliotropic papillomavirus. It encodes a transcriptional regulator, E2, which acts on the BPV-4 transcriptional control region (the long control region or LCR) to regulate transcription. The distribution of E2 binding sites within the LCR of BPV-4 is identical to that of the human papillomaviruses HPV-16 and HPV-18, indicating that the mechanism of transcriptional control by E2 of mucosal epitheliotropic papillomaviruses is conserved. In this study it has been shown that E2 activates transcription through the BPV-4 LCR promoter in primary bovine palate keratinocytes but not in primary bovine palate fibroblasts. The epithelial specific transcriptional activation of the BPV-4 LCR by E2 is promoter-specific because following binding to the BPV-4 LCR placed in an enhancer mode, E2 can activate transcription from heterologous promoters, such as SV40, in both keratinocytes and fibroblasts. Chimaeric VP16-E2 molecules suggest that the epithelial specific transcriptional activation of the BPV-4 LCR promoter is mediated by the E2 transactivation domain. Although low to intermediate levels of E2 can activate transcription from the BPV-4 LCR promoter, high levels of E2 result in down-regulation of transcription from this promoter in keratinocytes. Mutation of E2 binding site 1 (BS1), which is 3 bp upstream from the TATA box, abrogates down-regulation of transcription by high levels of E2. The results present a model system for studying transcriptional regulation of mucosal epitheliotropic papillomavirus LCRs by E2.

A peptide encoding a B-cell epitope from the N-terminus of the capsid protein L2 of bovine papillomavirus-4 prevents disease.

The first 200 N-terminus amino acids of the L2 capsid protein of BPV-4 (designated L2a) are an effective prophylactic vaccine against BPV-4 infection. Vaccination with L2a induces the production of virus neutralizing antibodies, and when L2a antibodies are removed from immune sera, the sera lose their neutralization activity. L2a encodes three dominant B-cell epitopes, defined as epitope 1 (amino acids 101-120), epitope 2 (aa 131-151), and epitope 3 (aa 151-170). To investigate whether any of these epitopes are responsible individually or in combination for protection against viral challenge, synthetic peptides, corresponding to the three epitopes (peptides 11, 14, and 16, respectively) and conjugated to keyhole limpet hemocyanin (KLH) were tested in vaccination challenge experiments. Calves vaccinated with the three peptides together showed no evidence of papillomavirus infection; those vaccinated with peptide 14 alone developed only early lesions which did not progress to proper papillomas and regressed rapidly; those vaccinated with peptide 11 or peptide 16 alone were not protected and proceeded to develop papillomas. Therefore the three B-cell epitopes are not conventionally "neutralizing" when presented individually, but in combination they form a complex neutralization domain, and, in particular, epitope 2, represented by peptide 14, encodes a domain responsible for disease prevention.

Viral proteins of bovine papillomavirus type 4 during the development of alimentary canal tumours.

In cattle infection of the upper alimentary canal mucosa by bovine papillomavirus type 4 (BPV-4) results in the development of papillomas which can progress to cancer in animals fed on bracken fern. This paper describes a study of the cellular and subcellular distribution of a number of different BPV-4 products in experimentally-induced BPV-4 tumours. E8 and E4 proteins were detected solely as cytoplasmic antigens in the undifferentiated and differentiated layers of the papilloma, respectively; L2 was detected solely as a nuclear antigen in the differentiated layers, whereas E7 was present in either the nucleus or the cytoplasm depending on the differentiation stage of the keratinocyte. Replicative forms of viral DNA were detected from the spinous to the squamous layers. Viral antigens were not detected during papilloma regression or in carcinomas. E8 was most prominent in early developmental stages, while E4 and L2 were most abundant in mature papillomas. E7 was present in large amounts in both early and mature stages, declining at later stages. These results suggest a temporal and spatial requirement for the expression and function of the viral proteins.

Linear B-cell epitopes in the N-terminus of L2 of bovine papillomavirus type 4.

The minor capsid protein L2 of bovine papillomavirus type 4 (BPV-4) is a very effective prophylactic vaccine which induces the production of virus neutralising antibodies and prevents virus-induced papillomatosis. The virus neutralising activity resides in the first 200 N-terminal amino acids of L2 (L2a). To further investigate the humoral immune response to L2, and the role it plays during infection and in prophylactic vaccination, the presentation of B-cell linear epitopes of L2a has been analysed in calves infected with the virus but not vaccinated, and in calves vaccinated with virus, L2a or E7. Several B-cell epitopes have been identified in L2a by the use of overlapping peptides; the epitopes varied in the different groups of animals, indicating that the epitopes presented by denatured L2 are not presented by the virus, and that therefore, although responsible for L2 vaccine-induced immunity, they may play little role in naturally acquired immunity.

Vaccination of cattle with bovine papillomavirus type 4 L2 elicits the production of virus-neutralizing antibodies.

Prophylactic vaccination of cattle with the N terminus (L2a, aa 11-200) of the minor capsid protein L2 completely prevented bovine papillomavirus type 4 (BPV-4) infection of the alimentary canal. To investigate the mechanisms underlying protection from viral infection, sera from vaccinated animals were analysed in neutralization assays both in the nude mouse xenograft system and in cattle. BPV-4 retained its infectivity when incubated with preimmune cattle sera, whereas, when incubated with immune sera from animals vaccinated with either whole L2 or its N terminus L2a, its infectivity was greatly reduced, indicating that the immune sera had neutralizing activity against the virus. This activity could be abrogated by absorbing the immune sera with L2 or L2a, thus indicating that virus neutralization was due to the presence in the immune sera of anti-L2 antibodies.

Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization.

Virus-like particles were produced in insect cells containing either the L1 and L2 capsid proteins of bovine papillomavirus type 4 (BPV-4) or only the L1 protein. Both preparations of VLPs proved to be extremely effective prophylactic vaccines. Thirteen of 15 calves immunised with either L1-L2 VLPs or L1-VLPs were refractory to experimental challenge with high doses of BPV-4 and did not develop papillomas, while 9 of 10 control animals developed multiple oral papillomas. VLPs were not efficient as therapeutic vaccine in calves with established papillomas, although VLP-vaccinated animals appeared to undergo tumour regression more rapidly than nonvaccinated control animals. Antibody responses in VLP-vaccinated calves were associated with prevention of disease but not with regression of papillomas. Thus prophylactic VLP vaccination is effective in preventing disease in this model of mucosal papillomavirus infection. VLPs and native virus share at least some conformational epitopes, as shown by the cross-reactivity of their antibodies.

Vaccination of cattle with the N-terminus of L2 is necessary and sufficient for preventing infection by bovine papillomavirus-4.

We have previously shown that cattle vaccinated with L2, the minor structural protein of bovine papillomavirus-4 (BPV-4), do not develop alimentary papillomas upon challenge with BPV-4. Analysis of the B and T cell response in L2-vaccinated animals showed that the majority of the response was directed against the N-terminus and C-terminus of L2 with little response against the middle portion. Cattle were vaccinated with the N-terminus or the C-terminus of L2. The animals vaccinated with the N-terminus were completely protected from viral challenge, whereas the animals vaccinated with the C-terminus were not. Further analysis with synthetic overlapping peptides spanning the entire N-terminus mapped a B cell immunodominant epitope at amino acid 101-120. This epitope was recognised by all vaccinated animals.

T cell responses to BPV-4 E7 during infection and mapping of T cell epitopes.

Vaccination of cattle with the recombinant E7 protein of bovine papillomavirus type 4 (BPV-4) prior to BPV-4 infection has been shown to retard development of papillomas and accelerate their regression. To understand the mechanism of regression we have measured proliferation of peripheral blood mononuclear cells (PBM) to E7 in vitro during the course of BPV-4 infection in both vaccinated and nonvaccinated cattle. In vaccinated cattle, T cells specific for E7 could be detected at high levels shortly after challenge, whereas in nonvaccinated cattle low responses of E7-specific T cells could be detected in only a few animals at the late stages of papilloma development. Using short overlapping synthetic peptides corresponding to the E7 protein, three T cell epitopes have been identified. T1 (aa 31-59) was immunodominant and T2 (aa 70-88) and T3 (aa21-40) were minor epitopes.

Humoral immune response to the E7 protein of bovine papillomavirus type 4 and identification of B-cell epitopes.

We have previously vaccinated cattle with E7, the major transforming protein of bovine papillomavirus-4, prior to homologous virus challenge. This retarded the development of papillomas and promoted their early regression compared to control animals. To understand the mechanism for this regression, we have studied the B and T cell response in vaccinated animals and compared it to that of non-vaccinated, virus-infected animals. The B cell response is reported here. The development of E7 IgG antibodies was detected after vaccination and before viral challenge, indicating that vaccine E7 is effectively presented to the immune system. In vaccinated animals titres of E7 antibodies remained high 10 weeks after viral challenge, whereas E7 antibodies in control animals were not detectable until 13 weeks post-viral challenge. Further analysis with synthetic overlapping peptides spanning the entire E7 protein mapped major immunodominant epitopes in the N and C termini of the protein and a minor epitope in the middle of the protein.

Prophylactic and therapeutic vaccination against a mucosal papillomavirus.

Papillomaviruses are ubiquitous DNA viruses affecting humans and animals and causing a variety of tumours of mucosal and cutaneous epithelia. Some of these lesions, particularly those affecting mucosal epithelia, can progress to squamous cell carcinomas. Prevention or cure of viral infection would ultimately lead to a decrease in the incidence of papillomavirus-associated cancers. Using recombinant proteins, we have developed prophylactic and therapeutic vaccines against bovine papillomavirus type 4, a mucosal papillomavirus implicated in cancer of the alimentary canal in cattle; similar possibilities exist for the human mucosal papillomaviruses.

Analysis of the transforming functions of bovine papillomavirus type 4.

Bovine papillomavirus type 4 (BPV-4) morphologically transforms primary bovine cells in vitro only in the presence of an activated ras gene. The transformed cells are capable of anchorage-independent growth, but are not immortal and are incapable of inducing tumors in nude mice. BPV-4 does not possess an E6 ORF and failure to induce full transformation may be due to the lack of this gene. Here we report the contribution of individual BPV-4 genes to cell transformation and the effect of adding the E6 ORF of HPV-16 to the system. We show that BPV-4 E7 ORF is responsible for morphological transformation, the E8 ORF is responsible for anchorage-independent growth, and addition of HPV-16 E6 ORF rescues cells from senescence. By immunocytostaining, E7 and E8 have been visualized in transiently transfected cultured cells. E8 is localized in the membranes while E7 is found both in the cytoplasm and in the nucleus.

Studies on vaccination against papillomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins.

The L1 and L2 proteins of BPV-2 have been produced in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins have been used to vaccinate calves both prophylactically and therapeutically. The L1 fusion protein prevented tumor formation when administered before challenge with BPV-2, while the L2 fusion protein was very effective in promoting tumor rejection, independently from whether it was administered before or after challenge. Animals vaccinated with L1, but not with L2, responded rapidly with production of serum neutralizing antibodies, showing that this peptide contains B-cell-specific epitopes. The massive infiltration of lymphocytes in the tumors of L2-vaccinated animals suggests that the peptide contains epitopes specific for T-cells. The two structural proteins of BPV-2 therefore interact with both efferent arms of the immune system, and this observation allows the choice between two different types of antiviral vaccination.

The B subgroup bovine papillomaviruses lack an identifiable E6 open reading frame.

Analysis of the corrected DNA sequence for the bovine papillomavirus type 4 (BPV4) genome revealed that there is no open reading frame (ORF) that might encode an E6 protein. The other two B subgroup bovine papillomaviruses, BPV3 and BPV6, were found to have the same arrangement of ORFs in this region as BPV4. Thus, we conclude that E6 functions are either not required by these viruses or are performed by another viral (or host) protein. Furthermore, the position that might be expected to be occupied by E6, between the long control region and the E7 ORF, contains the E8 ORF, which has the potential to encode a 42-residue polypeptide with considerable similarity to the E5 transforming protein of BPV1. Therefore, it appears that during the evolution of the B subgroup of BPVs, genomic rearrangements may have occurred resulting in the present layout of the early ORFs.

Interaction of epsilon-globin cis-acting control elements with erythroid-specific regulatory macromolecules.

We have used a competition assay to investigate the influence of erythroid-specific cellular factors on transcription from the human epsilon-globin major cap site promoter and the minor promoter located 200 base pairs (bp) upstream from the epsilon-globin cap site. In the human erythroid cell line K562, competition of the epsilon-globin major cap site promoter linked to the chloramphenicol acetyltransferase (CAT) gene (epsilon P-CAT) with the same promoter fragment linked to a neomycin resistance gene (epsilon P-NEO) leads to a reduction in CAT activity. This indicates the specific presence of K562 cells of factor(s) which interact with the 200-bp promoter fragment (isolated from the gene body or flanking sequences) to activate transcription from the epsilon-globin major cap site. Competition of the epsilon-globin major promoter (as epsilon P-CAT) with the upstream minor epsilon-globin promoter (as epsilon P2-NEO) also leads to a reduction in CAT activity, indicating that both promoters share erythroid-specific trans-acting factors. The reverse competition (epsilon P2-CAT with epsilon P-NEO) leads to an increase in CAT activity, suggesting that the existence of erythroid-specific factor(s) which repress transcription from the 200-bp-upstream epsilon-globin promoter.

Negative regulation of the human epsilon-globin gene by transcriptional interference: role of an Alu repetitive element.

The human epsilon-globin gene has a number of alternative transcription initiation sites which correspond with regions of DNase I hypersensitivity upstream of the canonical cap site. Transcripts originating from the promoters located -4.3/-4.5 and -1.48 kilobase pairs (kbp) and -900 and -200 base pairs (bp) upstream of the major epsilon-globin cap site can, at certain stages of erythroid differentiation, extend through the gene and are polyadenylated. The 350-bp PolIII transcripts, originating within the Alu repetitive element -2.2 kbp upstream of the cap site, extend in the opposite direction from the gene, are nonpolyadenylated, nucleus confined, and are detectable only in mature K562 cells or mature embryonic red blood cells where the epsilon-globin major cap site is maximally transcribed. Fragments containing the promoters located between -4.5 and -4.3 kbp upstream of the gene down regulate transcription from the epsilon-globin gene 20- to 30-fold in a transient expression assay in which both erythroid and nonerythroid cell lines were used. This occurs only when the direction of transcription from the -4.3/-4.5-kbp promoters is towards the gene, and we hypothesize that down regulation is caused by transcriptional interference. Fragments containing the Alu repetitive element -2.2 kbp upstream of the gene can overcome down regulation of the epsilon-globin gene by the -4.5-kbp element when interposed in the direct orientation between this element and the epsilon-globin gene.

Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562.

We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner.

Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes.

Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin.