Tumor progression in transgenic mice containing the bovine papillomavirus genome.
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Animals↗
Biomedical subjects
P M Howley
Publications and source records attributed to P M Howley.
The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex.
The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.
Animals↗
c-myc and pRB: role in TGF-beta 1 inhibition of keratinocyte proliferation.
The TGF-beta s are potent inhibitors of proliferation of most cell types in culture and in vivo. Previous studies have demonstrated that TGF-beta inhibition of skin keratinocyte proliferation involves suppression of c-myc transcription. Evidence derived from use of expression plasmids for certain DNA viral oncoproteins has suggested that the retinoblastoma gene (RB) may be involved in this process. Transient expression of pRB, like TGF-beta 1, in skin keratinocytes represses expression of a human c-myc reporter plasmid, and the same c-myc promoter region (TCE) is required for repression by either TGF-beta 1 or pRB. We showed here that proliferation and c-myc expression in a cell line lacking normal pRB (DU145 human prostate adenocarcinoma cells) are not inhibited by TGF-beta 1. Oligonucleotides containing the TCE were found to bind to a cellular protein of approximately 106 kD (termed p106) in Southwestern assays, utilizing extracts from both the skin keratinocytes and DU145 cells. TCE binding to p106 was diminished by TGF-beta in TGF-beta-sensitive skin keratinocytes but not in TGF-beta-insensitive SV40-transformed keratinocytes. These data support the hypothesis that pRB is required for TGF-beta 1 suppression of c-myc transcription and suggest the involvement of a cellular factor(s) in addition to pRB in the TGF-beta 1 pathway for inhibition of c-myc transcription and growth inhibition.
Animals↗
Oncoproteins encoded by the cancer-associated human papillomaviruses target the products of the retinoblastoma and p53 tumor suppressor genes.
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Amino Acid Sequence↗
Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA.
A methodologic study was performed to compare the polymerase chain reaction (PCR) and Southern blot hybridization, two commonly used testing strategies for the detection of human papillomavirus (HPV) infection. Three laboratories tested masked aliquots of exfoliated cervical cell specimens obtained from 120 women by cervicovaginal lavage. The study population included 32 women with condylomatous atypia or cervical intraepithelial neoplasia and 88 control women with no known history of cervical neoplasia. Two laboratories used PCR with different sets of consensus primers for HPV detection. The third laboratory used low-stringency Southern blot hybridization to identify all HPV types, followed by high-stringency Southern and/or dot blot hybridization to confirm specific HPV types. One of the PCR primer sets detected HPV types with a differential efficiency that was not predicted by analysis of DNA sequences or direct testing of HPV-containing plasmids. In contrast, the second PCR primer set was shown to be a much broader consensus system, detecting the same HPV types as Southern blotting, though requiring much less clinical specimen. Over 80% of women with cervical intraepithelial neoplasia or condylomatous atypia were found to be HPV infected both by Southern blotting and by the second PCR primer set. Among the control women, 11% were HPV positive by Southern blotting, while 31% were positive with the second set of primers. Most of the HPV infections found only by PCR were not due to HPV type 6, 11, 16, 18, 31, 33, or 45. These known HPV types were uncommon among normal women in the study population, even as determined by the PCR method.
Base Sequence↗
Bovine papillomavirus with a mutation in the E2 serine 301 phosphorylation site replicates at a high copy number.
The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes at least three proteins with transcriptional regulatory properties. The full-length E2 open reading frame encodes a transcriptional transactivator, and the 3' region encodes two smaller polypeptides that repress E2-mediated transactivation. The full-length gene product is also required for viral DNA replication. We have demonstrated that the BPV-1 E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the carboxy-terminal DNA binding domain, which is common to all three E2 proteins (A. A. McBride, J. B. Bolen, and P. M. Howley, J. Virol. 63:5076-5085, 1989). These serine residues, at amino acid positions 298 and 301, were substituted with alanine residues in the context of the entire BPV-1 genome. The mutated BPV-1 genomes were introduced into rodent cell lines and assayed for focus formation, viral gene expression, and extrachromosomal viral DNA replication. Viral DNAs containing the E2 serine-to-alanine substitution mutants transformed C127 cells with efficiencies comparable to that of wild-type BPV-1. However, the viral genome containing the serine-to-alanine substitution at position 301 of the E2 polypeptide replicated to a copy number 20-fold higher than that of wild-type DNA.
Amino Acid Sequence↗
Analysis of trans activation by human papillomavirus type 16 E7 and adenovirus 12S E1A suggests a common mechanism.
The human papillomavirus E7 gene product is an oncoprotein with properties similar to those of the adenovirus E1A proteins. The human papillomavirus E7 proteins possess substantial amino acid sequence similarity to portions of conserved regions 1 and 2 of E1A, and the human papillomavirus type 16 E7 protein trans-activates the adenovirus E2 early promoter. Analysis of point mutations in the E2 promoter indicated that the E2F recognition sites were critical to E7 stimulation. In contrast to the activation of the E2 promoter, E7 could not trans-activate various other E1A-inducible promoters. Although the promoter specificity for E7 differs from that of 13S E1A trans activation, it is very similar to activation by the E1A 12S product. Moreover, analysis of the E7 protein has suggested that amino acid sequences critical for trans activation include those shared with E1A within conserved region 2. Biochemical studies demonstrate that the E7 protein, like the 12S E1A product, can alter the interaction of cellular factors with the E2F transcription factor. We therefore conclude that E7 trans activation is functionally related to that mediated by the 12S E1A product.
Adenovirus Early Proteins↗
Characterization of the cis elements involved in basal and E2-transactivated expression of the bovine papillomavirus P2443 promoter.
Transcriptional transactivation and repression by the viral E2 proteins are important regulatory mechanisms for the papillomaviruses. In the bovine papillomavirus type 1 (BPV-1), several viral promoters can be transactivated by E2 through E2-dependent enhancer elements located in the viral long control region (LCR), including promoters involved in E2 expression itself. This report demonstrates that the BPV-1 P2443 promoter is transactivated by E2-responsive elements in the LCR and that this promoter is responsible for a major part of the expression of the E2 and E5 gene products. Characterization of the cis elements involved in P2443 regulation indicated that the single E2-binding site directly upstream of P2443 is not required for either the E2 transactivation or for any E2 repression of the basal or transactivated activity of this promoter. Therefore, cooperative interactions between E2 bound at the LCR and E2 bound near P2443 do not have any role in the regulation of this promoter. Further definition of the cis regulatory elements of this promoter indicated that a binding site for the transcriptional factor Sp1 exists directly upstream of the P2443 TATA box and is critical for the basal level of transcription from this promoter. Disruption of this Sp1 site eliminated P2443 promoter activity in transient expression assays for E2 and E5 and resulted in a loss of transforming activity when introduced into the full viral genome.
Animals↗
The viral transcriptional regulatory region upstream of the E6 and E7 genes is a major determinant of the differential immortalization activities of human papillomavirus types 16 and 18.
The human papillomavirus types 16 (HPV-16) and 18 (HPV-18) can immortalize primary human keratinocytes. The region of the viral genome responsible for this function maps to the E6 and E7 genes and their respective upstream transcriptional regulatory sequences, the long control regions (LCRs). The HPV-18 LCR/E6/E7 is more efficient in this immortalization function than the analogous region of the HPV-16 genome, resembling the difference in the immortalization potentials of the two full-length viral genomes. This study was designed to examine the basis for the difference in HPV-16 and HPV-18 immortalization efficiencies. The E6 and E7 genes of either HPV-16 or HPV-18, when expressed from the same heterologous promoter, immortalized primary human keratinocytes with the same low efficiency, suggesting that the difference in immortalization activities was not due to the different E6 or E7 genes themselves but rather to a difference in the transcriptional regulatory regions upstream of these genes. The analysis of a series of chimeric HPV-16 and HPV-18 LCR/E6/E7 constructs confirmed this observation and further mapped the viral element responsible for the major difference in immortalization efficiency to the transcriptional regulatory region upstream of the E6 and E7 genes.
Base Sequence↗
Biochemical and biological differences between E7 oncoproteins of the high- and low-risk human papillomavirus types are determined by amino-terminal sequences.
Differences in the biological characteristics of the high-risk human papillomavirus type 16 (HPV-16) and the low-risk HPV-6 E7 proteins were analyzed and shown to correlate with certain biochemical properties. To ascertain which region of E7 conferred these properties, chimeric E7 genes were constructed by the exchange of the amino and carboxyl coding halves of the HPV-6 and HPV-16 E7 genes. The amino-terminal half of E7 determined the affinity for binding to the retinoblastoma protein pRB, the transformation properties, and the ability to abrogate transforming growth factor beta-mediated repression of the c-myc promoter. This region of E7 is therefore responsible for the biological and biochemical differences between the E7 proteins of the low-risk and the high-risk HPVs and consequently is one of the critical determinants distinguishing these two groups of viruses. Transcriptional transactivation of the adenovirus E2 promoter, in contrast, was a property shared by E7 proteins of both low-risk and high-risk HPVs.
Adenovirus Early Proteins↗
Functional analysis of E2-mediated repression of the HPV18 P105 promoter.
Transcription of the transforming genes E6 and E7 of the human papillomavirus type 18 (HPV18) can be repressed by the product of the E2 open reading frame. Mutations were introduced in the cis-responsive elements upstream from the E6 and E7 transcriptional promoter, P105. The effect of these mutations in the absence or presence of E2 was examined in the human cervical carcinoma cell line C33, transfected with expression plasmids. In the presence of HPV18 E2, repression was relieved only when both of the two E2 binding motifs, immediately proximal to the P105 TATA box, were mutated. Mutation of a third E2 binding site, 110 nucleotides upstream of the TATA box, led to a slight but consistent activation in the presence of the full-length E2. Therefore, three E2 binding sites appear to be involved in the negative regulation of the P105 promoter by HPV18 E2. In the presence of the BPV1 E2 protein, only the E2 binding site most proximal to the promoter TATA box was involved in the P105 repression. The comparative analysis of the in vitro and in vivo properties of the human and bovine E2 proteins on the HPV18 P105 promoter provides new insights into the mechanism of transcriptional repression, which appears to involve steric hindrance at the site of transcriptional initiation.
Base Sequence↗
Role of the human papillomavirus oncoproteins in transformation and carcinogenic progression.
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Amino Acid Sequence↗
Cellular targets of the oncoproteins encoded by the cancer associated human papillomaviruses.
Insight into the mechanisms by which DNA tumor viruses transform cells has come from the recognition that the virus-encoded oncoproteins interact specifically with important cell regulatory proteins. The "high risk" human papillomaviruses such as HPV-16 and HPV-18 which are associated with human anogenital carcinomas encode two transforming genes (E6 and E7) which are expressed in HPV positive cancers and derived cell lines. E7 shares functional and structural features with the adenovirus E1A proteins. Like Ad E1A and the large T proteins of the polyomaviruses, E7 can complex pRB. The E7 proteins of the "high risk" HPVs associate with pRB with approximately a 10-fold higher affinity than do the E7 proteins of the "low risk" HPVs, and important biological differences between the E7 proteins of these two groups of HPVs are determined by amino-terminal sequences which include the pRB binding domain. Like SV40 large T and Ad 5 E1B, the E6 oncoprotein encoded by the "high risk" HPVs can form a complex with p53. In vitro, E6 promotes the degradation of p53 and this degradation involves the ubiquitin-dependent protease system. The selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant acting oncoproteins. The relevance of the inactivation of the normal functions of pRB and p53 in human cervical carcinogenesis has recently been demonstrated by the analysis of these two genes and their products in a series of HPV-positive and HPV-negative cell lines. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild type p53 proteins, which are presumed to be altered in function as a consequence of association with the HPV oncoproteins. In contrast, mutations were identified in the p53 and RB genes expressed in the HPV-negative cervical carcinoma cell lines, C33-A and HT-3. These results support the hypothesis that the inactivation of the normal functions of the tumor suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or through complex formation with HPV E6 and E7 oncoproteins.
Cell Line↗
The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.
The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.
Antigens, Polyomavirus Transforming↗
TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains.
TGF-beta 1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-beta 1. Since TGF-beta 1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-beta 1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-beta 1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-beta 1 regulation of c-myc gene expression and growth inhibition.
Adenovirus Early Proteins↗
Association of human papillomavirus types 16 and 18 E6 proteins with p53.
Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.
Adenovirus Early Proteins↗
A bovine papillomavirus constitutive enhancer is negatively regulated by the E2 repressor through competitive binding for a cellular factor.
The bovine papillomavirus type 1 long control region (LCR) contains DNA sequence elements involved in the regulation of viral transcription and replication. Differences in the levels of transcription have previously been noted between bovine papillomavirus type 1-infected rodent cell lines and bovine cells. To investigate these differences, fragments of the LCR were cloned into an enhancer-deleted chloramphenicol acetyltransferase expression vector and assayed for enhancer activity. A strong constitutive enhancer was found in the 5' portion of the LCR that was most active in primary bovine fibroblasts and had little activity in other cell types. Deletion mapping localized most of the activity to a 113-bp fragment from nucleotides (nt) 7162 to 7275, a region of the viral sequence that also contains the P7185 promoter and an E2-binding site at nt 7203. The enhancer activity of this element could be positively modulated by the full-length E2 transactivator or negatively modulated by the E2 repressor. Site-directed mutagenesis defined two cis elements, CE1 and CE2, which were both necessary for enhancer activity. The CE1 element was required for P7185 activity, whereas the CE2 element was dispensable for P7185 activity. The CE1 and CE2 elements both overlap the E2-binding site at nt 7203. In vitro DNA-binding studies revealed (i) a specific gel retardation complex associated with cellular factor binding at the CE1 element, (ii) a correlation between enhancer activity and the binding of factors to the CE1 element, and (iii) competitive binding between the E2 repressor and the cellular factor at the CE1 element.
Animals↗
Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants.
The bovine papillomavirus type 1 (BPV-1) E2 open reading frame encodes three proteins: the E2 transcriptional transactivator, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion peptide. In this study, we describe the phenotypes of BPV-1 mutants which are disrupted in their capacity to encode either the E2 transcriptional repressor or the E8/E2 fusion peptide. We also describe experiments which demonstrate that the E8/E2 gene product functions similarly to E2-TR. In the context of the entire viral genome, disruption of E8/E2 expression had little effect on viral processes, whereas disruption of E2-TR expression resulted in a complex phenotype marked by a 10- to 20-fold increase in viral DNA plasmid copy number as well as increased transformation potential. A double mutant, defective in the expression of both E8/E2 and E2-TR proteins, had high levels of E2 transactivation activity yet had reduced plasmid replication capacity and a delayed capacity to transform rodent cells.
Animals↗