Differentiation-specific expression from the bovine papillomavirus type 1 P2443 and late promoters.

The papillomavirus life cycle is tightly linked with keratinocyte differentiation in squamous epithelia. Vegetative viral DNA replication begins in the spinous layer, while synthesis of capsid proteins and virus maturation is restricted to the most differentiated or granular layer of the epithelium. In this study, in situ hybridization of bovine fibropapillomas was used to demonstrate that the activity of two promoters of bovine papillomavirus type 1 (BPV-1) is regulated in a differentiation-specific manner. In situ hybridization with a late promoter (PL)-specific oligonucleotide probe suggested that PL is dramatically upregulated in the granular layer of the fibropapilloma. Northern (RNA) blot analysis of RNA from BPV-1-infected fibropapillomas indicated that the three major BPV-1 late-region mRNAs were transcribed from PL. These RNAs include the previously described L1 (major capsid) mRNA as well as two larger mRNAs. The two larger mRNAs were characterized and shown to contain the L2 (minor capsid protein) open reading frame as well as the L1 open reading frame. In contrast to PL, the P2443 promoter was maximally active in basal keratinocytes and the fibroma. The major mRNA transcribed from P2443 is the putative E5 oncoprotein mRNA which is spliced between nucleotides 2505 and 3225. No signal was detected above the basal layer with use of a probe specific for this mRNA. The E5 oncoprotein has previously been localized by immunoperoxidase staining to the granular cell layer as well as the basal cell layer of the fibropapilloma (S. Burnett, N. Jareborg, and D. DiMaio, Proc. Natl. Acad. Sci. USA 89:5665-5669, 1992). These data suggest that E5 proteins in the basal cell and granular cell layers are not translated from the same mRNA.     

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.     

Rb-independent induction of apoptosis by bovine papillomavirus type 1 E7 in response to tumor necrosis factor alpha

Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor alpha (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A(2) was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB.     

Interaction of the Bovine Papillomavirus E6 Protein with the Clathrin Adaptor Complex AP-1

The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the γ subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.     

The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.     

Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus

Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.     

S100 protein subcellular localization during epidermal differentiation and psoriasis.

S100 proteins are calcium-activated signaling proteins that interact with target proteins to modulate biological processes. Our present studies compare the level of expression, and cellular localization of S100A7, S100A8, S100A9, S100A10, and S100A11 in normal and psoriatic epidermis. S100A7 and S100A11 are present in the basal and spinous layers in normal epidermis. These proteins appear in the nucleus and cytoplasm in basal cells but are associated with the plasma membrane in spinous cells. S100A10 is present in basal and spinous cells, in the cytoplasm, and is associated with the plasma membrane. S100A8 and S100A9 are absent or are expressed at minimal levels in normal epidermis. In involved psoriatic tissue, S100A10 and S100A11 levels remain unchanged, whereas, S100A7, S100A8, and S100A9 are markedly overexpressed. The pattern of expression and subcellular localization of S100A7 is similar in normal and psoriatic tissue. S100A8 and S100A9 are strongly expressed in the basal and spinous layers in psoriasis-involved tissue. In addition, we demonstrate that S100A7, S100A10, and S100A11 are incorporated into detergent and reducing agent-resistant multimers, suggesting that they are in vivo transglutaminase substrates. S100A8 and S100A9 did not form these larger complexes. These results indicate that S100 proteins localize to the plasma membrane in differentiated keratinocytes, suggesting a role in regulating calcium-dependent, membrane-associated events. These studies also indicate, as reported previously, that S100A7, S100A8, and S100A9 expression is markedly altered in psoriasis, suggesting a role for these proteins in disease pathogenesis.     

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.     

Bovine papillomavirus E7 oncoprotein inhibits anoikis

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

Bovine papillomavirus oncoprotein E5 affects the arachidonic acid metabolism in cells

The bovine papillomavirus type 1 (BPV-1) oncoprotein encoded by the E5 ORF is a small highly hydrophobic protein, which is capable of inducing oncogenic transformation of cells. We studied the effect of the BPV-1 E5 protein expression on the arachidonic acid metabolism in monkey (COS1) and human (C33A) cells. At relatively low protein concentrations the phospholipase A(2) (PLA(2)) activity and the arachidonic acid (AA) metabolism are activated. E5 mutant proteins, lacking cysteines responsible for the dimerisation of the protein (C37S, C37SC39S), and truncated E5, lacking the C-terminal region, are non-transforming and unable to stimulate the PLA(2) activity and AA metabolism. The transformation-defective mutant D33V, which does not activate the platelet-derived growth factor receptor (PDGFR), activates AA metabolism like wt E5. Our data suggest that the BPV-1 E5 protein could stimulate the AA metabolism independently of PDGF receptor.     

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo.     

Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996