Biomedical subjects

M S Campo

Publications and source records attributed to M S Campo.

At least 19 recordsLinked to original sources

PubMed2006

The E5 oncoprotein of BPV-4 does not interfere with the biosynthetic pathway of non-classical MHC class I.

The major histocompatibility complex (MHC) class I region in mammals contains both classical and non-classical MHC class I genes. Classical MHC class I molecules present antigenic peptides to cytotoxic T lymphocytes, whereas non-classical MHC class I molecules have a variety of functions. Both classical and non-classical MHC molecules interact with natural killer cell receptors and may under some circumstances prevent cell death by natural killer cytotoxicity. The E5 oncoprotein of BPV-4 down-regulates the expression of classical MHC class I on the cell surface and retains the complex in the Golgi apparatus. The inhibition of classical MHC class I to the cell surface results from both the impaired acidification of the Golgi, due to the interaction of E5 with subunit c of the H+ V-ATPase, and to the physical binding of E5 to the heavy chain of MHC class I. Despite the profound effect of E5 on classical MHC class I, E5 does not retain a non-classical MHC class I in the Golgi, does not inhibit its transport to the cell surface and does not bind its heavy chain. We conclude that, as is the case for HPV-16 E5, BPV-4 E5 does not down-regulate certain non-classical MHC class I, potentially providing a mechanism for the escape of the infected cell from attack by both cytotoxic T lymphocytes and NK cells.

Animals↗

PubMed2006

Down-regulation of MHC class I is a property common to papillomavirus E5 proteins.

The E5 protein family of papillomaviruses comprises small hydrophobic proteins which are associated with the cell endomembrane compartments. The functions of the E5 proteins, particularly those of HPV, are still far from clear. We have reported that the E5 proteins of BPV-1, BPV-4, HPV-16 and HPV-6 down-regulate MHC class I, potentially helping the virus evade the host immune response. Others have described MHC class I down-regulation by HPV-2 E5. We report here that another E5 protein, HPV-83 E5, likewise down-regulates MHC class I and propose that interference with expression, assembly and/or transport of MHC class I is a common property of all E5 proteins evolved by the virus to circumvent host immunosurveillance and thus establish productive infection.

Cell Line↗

PubMed2006

The E5 protein of BPV-4 interacts with the heavy chain of MHC class I and irreversibly retains the MHC complex in the Golgi apparatus.

BPV-4 E5 inhibits transcription of the bovine MHC class I heavy chain (HC) gene, increases degradation of HC and downregulates surface expression of MHC class I by retaining the complex in the Golgi apparatus (GA). Here we report that transcription inhibition can be alleviated by interferon treatment and the degradation of HC can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC class I to the cell surface is irreversible. We show that E5 is capable of physically interacting with HC. Together with the inhibition of the vacuolar ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and HC is likely to be responsible for retention of MHC class I in the GA. C-terminus deletion mutants of E5 are incapable of either downregulating surface MHC class I or interacting with HC, establishing that the C-terminus domain of E5 is important in the inhibition of MHC class I.

Animals↗

PubMed2006

Bovine papillomavirus E5 oncoprotein binds to the activated form of the platelet-derived growth factor beta receptor in naturally occurring bovine urinary bladder tumours.

Studies regarding the functions of the bovine papillomavirus (BPV) E5 oncoprotein in vivo are lacking and no E5-mediated mechanism underlying epithelial carcinogenesis is known. We have shown that BPV-2 DNA is present in the majority of naturally occurring urinary bladder tumours of cattle and that E5 is expressed in the cancer cells. Here we show that the interaction between the platelet-derived growth factor (PDGF) beta receptor and BPV E5, described in vitro in cultured cells, takes place in vivo in bovine urinary bladder cancers. In these cancers, E5 and PDGF beta receptor colocalize, as shown by confocal microscopy, and physically interact, as shown by coimmunoprecipitation. Furthermore, the PDGF beta receptor associated with E5 is highly phosphorylated, suggesting the functional activation of the receptor upon E5 interaction. Our results demonstrate, for the first time, that E5-PDGF beta receptor interaction occurs during the natural history of bovine urinary bladder tumours, suggesting an important role for E5 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E5. Successful bladder tumour regression would provide a valuable model for therapeutic vaccination against papillomavirus-associated tumours.

Adenocarcinoma↗

PubMed2005

HPV-18 transformed cells fail to arrest in G1 in response to quercetin treatment.

Previous work with primary human keratinocytes demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 with concomitant elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins, under transcriptional control of a heterologous promoter, in transformed keratinocytes failed to abrogate this arrest [Beniston, R., Campo, M.S., 2003. Quercetin elevates p27Kip1 and arrests both primary and HPV-16 E6/E7 transformed human keratinocytes in G1. Oncogene 22, 5504-5514]. Given the link between papillomavirus infection, bracken fern in the diet and cancer of the oesophagus in humans, we wished to investigate further whether cells transformed by the whole genome of HPV-16 or HPV-18, with E6 and E7 under the transcriptional control of their respective homologous promoters, would be similarly arrested in G1 by quercetin. In agreement with earlier work, quercetin arrested HPV-16 transformed cells in G1 with an increase in the cyclin-dependent kinase inhibitor p27Kip1. However, HPV-18 transformed cells did not arrest after quercetin treatment. The failure of HPV-18 transformed cells to arrest in G1 was linked to the up-regulation of the HPV-18 long control region (LCR) by quercetin, maintaining high expression of the viral transforming proteins. Transcriptional up-regulation of the HPV-18 LCR was mediated by a "quercetin responsive element" homologous to the one identified previously in the bovine papillomavirus type 4 (BPV-4) LCR.

Cell Cycle Proteins↗

PubMed2005

Human papillomavirus 16 L2 inhibits the transcriptional activation function, but not the DNA replication function, of HPV-16 E2.

In this study we analysed the outcome of the interaction between HPV-16 L2 and E2 on the transactivation and DNA replication functions of E2. When E2 was expressed on its own, it transactivated a number of E2-responsive promoters but co-expression of L2 led to the down-regulation of the transcription transactivation activity of the E2 protein. This repression is not mediated by an increased degradation of the E2 protein. In contrast, the expression of L2 had no effect on the ability of E2 to activate DNA replication in association with the viral replication factor E1. Deletion mutagenesis identified L2 domains responsible for binding to E2 (first 50 N-terminus amino acid residues) and down-regulating its transactivation function (residues 301-400). The results demonstrate that L2 selectively inhibits the transcriptional activation property of E2 and that there is a direct interaction between the two proteins, although this is not sufficient to mediate the transcriptional repression. The consequences of the L2-E2 interaction for the viral life cycle are discussed.

Capsid Proteins↗

PubMed2004

Downregulation of major histocompatibility complex class I in bovine papillomas.

Bovine papillomavirus (BPV) induces papillomas in cattle; in the great majority of cases, these regress due to the host immune response, but they can persist and progress to malignancy. Even in the absence of malignant transformation, BPV infection persists for a significant period of time before activation of the host immune system, suggesting that the host immune system is unaware of, or disabled by, BPV. E5 is the major oncoprotein of BPV, which, in addition to its transforming properties, downregulates the expression and transport to the cell surface of major histocompatibility complex class I (MHC I). Here, it is shown that co-expression of MHC I and E5 in papillomas caused by BPV-4 infection is mutually exclusive, in agreement with the inhibition of surface MHC I expression by E5 that is observed in vitro. The inhibition of MHC expression in E5-expressing papilloma cells could explain the long period that is required for activation of the immune response and has implications for the progression of papillomas to the malignant stage; absence of peptide presentation by MHC I to cytotoxic T lymphocytes would allow the infected cells to evade the host cellular immune response and allow the lesions to persist.

Animals↗

PubMed2003

Sequence variants of bovine papillomavirus E5 detected in equine sarcoids.

The equine sarcoid, one of the most common dermatological lesions in equids, is a benign, locally invasive dermal fibroblastic lesion. Previous studies have suggested an association with two bovine papilloma virus (BPV) types, BPV-1 and BPV-2. In the present study, we examined sarcoids from horses from two geographical areas, Switzerland and the UK, for the major transforming gene of BPV, E5. We detected BPV DNA for the E5 open reading frame and viral E5 RNA transcripts in most sarcoids. Sequence analysis of the E5 open reading frame of sarcoid-associated BPV detected several unique DNA sequence variants, three of which resulted in sarcoid specific amino acid sequence variations. It is unclear if these sequence variants contribute to the unique clinical presentation of the sarcoid. However, our work provides further evidence of the association between BPV and sarcoid development and the direct involvement of the virus in the pathogenesis of sarcoids.

Animals↗

PubMed2003

Association of bovine papillomavirus with the equine sarcoid.

The equine sarcoid, a locally aggressive, fibroblastic skin tumour, is the most common dermatological neoplasm reported in horses; there is no consistently effective therapy. It is widely accepted that bovine papillomavirus (BPV) types 1 and 2 are associated with the pathogenesis of sarcoid disease. Most sarcoids appear to contain detectable viral DNA and RNA and are also known to express the BPV types 1 and 2 major transforming protein, E5, but appear not to produce infectious virions. While the mode of transmission of infection has not been elucidated, viral gene expression, in particular of E5, may contribute to virus persistence and disease pathogenesis by downregulating MHC class I expression. Here, the pathology and epidemiology of the sarcoid and its association with BPV is reviewed; the transforming functions of the BPV oncoproteins and their possible role in sarcoid pathogenesis are discussed; and the practical implications of BPV infection for diagnostic and therapeutic purposes are considered.

Animals↗

PubMed2001

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.

3T3 Cells↗

PubMed2001

Immunoglobulin genes expressed by B-lymphocytes infiltrating cervical carcinomas show evidence of antigen-driven selection.

Lymphocyte infiltration is often present in cervical cancer lesions, possibly reflecting an ongoing (but ineffective) immune response to the tumour. B-lymphocytes are the predominant lymphocyte infiltrate in pre-malignant cervical lesions, where they are thought to comprise the host immune response to active human papillomavirus (HPV) infection. Although B cells are less frequently detected in cervical tumours, a high proportion of terminally differentiated plasma cells expressing tumour-specific immunoglobulin (Ig) remain. The antigen specificity and functional significance of the antibody response to cervical tumours is unknown. As part of a study to characterise the antibodies expressed by the tumour-infiltrating B cells (TIL-B) in cervical tumours using antibody phage display, we examined expressed Ig gene sequences to determine if there was molecular evidence of a selective response to antigenic changes in the transformed epithelial cells. We found that biased variable region gene usage by the B cells and the rate of somatic hypermutation in the rearranged Ig heavy chain variable regions (VH) both indicated antigenic selection of the B cells. We also found evidence of affinity maturation, as indicated by the detection of antibodies of the IgG1, IgG2 and IgA isotypes, and possible clonal selection of the Ig receptors. These data support the notion that B-lymphocytes and plasma cells infiltrating cervical carcinomas are the result of an antigen-induced response to HPV infection or transformation.

Antigen Presentation↗

PubMed2001

Quercetin, E7 and p53 in papillomavirus oncogenic cell transformation.

Bovine papillomavirus type 4 (BPV-4) infects the upper alimentary canal of cattle causing benign papillomas which can progress to squamous carcinomas in cattle grazing on bracken fern (BF). We have previously shown that quercetin, a well characterized and potent mutagen found in BF, causes cell cycle arrest of primary bovine cells (PalF), but that a single exposure to quercetin can cause full oncogenic transformation of PalF cells partially transformed by BPV-4. Here we show that cell cycle arrest correlates with an increase in p53 protein levels and transcriptional activity. However, in cells transformed but non-tumorigenic, p53 protein is elevated and transcriptionally activated in response to quercetin or other DNA damaging stimuli, but the cells bypass quercetin-induced G1 arrest likely due to E7 expression. In transformed tumorigenic cells, p53 is elevated in response to quercetin but its transcriptional activity is inhibited due to mutation, and the cells fail to stop in G1 in the presence of quercetin.

Base Sequence↗

PubMed2001

A novel silencer element in the bovine papillomavirus type 4 promoter represses the transcriptional response to papillomavirus E2 protein.

The long control regions (LCRs) of mucosal epitheliotropic papillomaviruses have similar organizations: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites (C. Desaintes and C. Demeret, Semin. Cancer Biol. 7:339--347, 1996). The enhancer of these viruses is epithelial cell specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (B. Gloss, H. U. Bernard, K. Seedorf, and G. Klock, EMBO J. 6:3735--3743, 1987). Using the bovine papillomavirus type 4 (BPV-4) LCR and a bovine primary cell system, we have shown previously that a level of epithelial specificity resides in a papillomavirus promoter region. The BPV-4 promoter shows an enhanced response to transcriptional activators in epithelial cells compared with that of fibroblasts (K. W. Vance, M. S. Campo, and I. M. Morgan, J. Biol. Chem. 274:27839--27844, 1999). A chimeric lcr/tk promoter suggests that the upstream BPV-4 promoter region determines the cell-type-selective response of this promoter in fibroblasts and keratinocytes. Promoter deletion analysis identified two novel repressor elements that are, at least in part, responsible for mediating the differential response of this promoter to upstream activators in fibroblasts and keratinocytes. One of these elements, promoter repressor element 2 (PRE-2), is conserved in position and sequence in the related mucosal epitheliotropic papillomaviruses, BPV-3 and BPV-6. PRE-2 functions in cis to repress the basal activity of the simian virus 40 promoter and binds a specific protein complex. We identify the exact nucleotides necessary for binding and correlate loss of binding with loss of transcriptional repression. We also incorporate these mutations into the BPV-4 promoter and demonstrate an enhanced response of the mutated promoter to E2 in fibroblasts. The DNA binding protein in the detected complex is shown to have a molecular mass of approximately 50 kDa. The PRE-2 binding protein represents a novel transcriptional repressor and regulator of papillomavirus transcription.

Animals↗

PubMed2000

alpha6 integrin is not the obligatory cell receptor for bovine papillomavirus type 4.

Recently, alpha6 integrin has been proposed as the epithelial cell receptor for papillomavirus. This study investigated whether alpha6 integrin is the cellular receptor for bovine papillomavirus type 4 (BPV-4), which is strictly epitheliotropic and infects the mucous epithelium of the upper digestive tract. Primary bovine mucosal keratinocytes from the palate of a foetus (PalK) displayed high levels of alpha6 integrin; matched primary fibroblasts from the same biopsy (PalF) expressed almost no alpha6 integrin. However, BPV-4 bound both PalK and PalF to similar, saturable levels. Native BPV-4 virions infected PalK in vitro, as detected by RT-PCR of E7 RNA. Infection could be blocked by excess virus-like particles (VLPs) and by neutralizing antisera against L1-L2 and L1 VLPs or by denaturation of the virions, supporting the view that infection in vitro mimics the process in vivo. alpha6 integrin-negative human keratinocyte cell lines were derived from patients affected by junctional epidermolysis bullosa presenting genetic lesions in their hemidesmosomes. The level of alpha6 integrin expression was determined in these cell lines by in situ immunofluorescence and FACS. Despite the absence of alpha6 integrin expression by BO-SV cells, they were bound by BPV-4 to similar, saturable levels as normal keratinocytes, KH-SV. Furthermore, BO-SV and KH-SV cells were both infected by BPV-4 to apparently the same extent as PalK cells. These results are consistent with the conclusion that alpha6 integrin is not the obligatory receptor for a bovine mucosotropic papillomavirus.

Animals↗

PubMed1999

An enhanced epithelial response of a papillomavirus promoter to transcriptional activators.

Mucosal epitheliotropic papillomaviruses have a similar long control region (LCR) organization: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites. The enhancer of these viruses is epithelial-specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (Gloss, B., Bernard, H. U., Seedorf, K., and Klock, G. (1987) EMBO J. 6, 3735-3743; Morgan, I. M., Grindlay, G. J., and Campo, M. S. (1999) J. Gen. Virol. 80, 23-27). Studies on E2 transcriptional regulation of the human mucosal epitheliotropic papillomaviruses have been hindered by poor access to the natural target cell type and by the observation that some of the human papillomavirus promoters, including human papillomavirus-16, are repressed in immortalized epithelial cells. Here we present results using the bovine papillomavirus-4 (BPV-4) LCR and a bovine primary cell system as a model to study the mechanism of E2 transcriptional regulation of mucosal epitheliotropic papillomaviruses and the cell type specificity of this regulation. E2 up-regulates transcription from the BPV-4 LCR preferentially in epithelial cells (Morgan, I. M., Grindlay, G. J., and Campo, M. S. (1998) J. Gen. Virol. 79, 501-508). We demonstrate that the epithelial-specific enhancer element of the BPV-4 LCR is not required for the enhanced activity of E2 in epithelial cells and that the BPV-4 promoter is more responsive, not only to E2, but to other transcriptional activators in epithelial cells. This is the first time a level of epithelial specificity has been shown to reside in a papillomavirus promoter region.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Generation of native bovine mAbs by phage display.

Modeling of disease pathogenesis and immunity often is carried out in large animals that are natural targets for pathogens of human or economic relevance. Although murine mAbs are a valuable tool in identifying certain host/pathogen interactions, progress in comparative immunology would be enhanced by the use of mAbs isolated from the host species. Such antibodies would reflect an authentic host immune response to infection or vaccination, and as they are host derived, would allow the application of in vivo experiments that previously have been unrealizable in large animals because of induction of an antispecies immune response. The advent of antibody phage display technology provides a way of producing host-derived mAbs in animals where the molecular genetics of Ig formation are known. Exploiting recent advances in the molecular immunology of cattle, we report here the design of an optimized phage display vector, pComBov, for the construction of combinatorial libraries of bovine Ig antigen-binding fragments (Fab) of native sequence. By using this system, we initially have generated and characterized a panel of bovine mAbs against a model antigen glutathione S-transferase. The isolated mAbs showed features typical of bovine Igs and recognized glutathione S-transferase with high specificity in ELISA and by Western blotting. The pComBov expression system can be readily adapted for the preparation of libraries from related ruminant species and advances the use of monoclonal reagents derived in this way for comparative studies in animals of economic importance.

Amino Acid Sequence↗

PubMed1999

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.

Animals↗