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P M Howley

Publications and source records attributed to P M Howley.

At least 109 records · Page 6Linked to original sources
PubMed1990

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.

Base Sequence↗
PubMed1989

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.

Amino Acid Sequence↗
PubMed1989

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

Adenovirus Early Proteins↗
PubMed1989

Specific chromosomal abnormalities characterize fibrosarcomas of bovine papillomavirus type 1 transgenic mice.

In the BPV1.69 line of transgenic mice, the bovine papillomavirus type 1 genome elicits both benign dermal fibroblastic proliferation (fibromatoses) and malignant fibrosarcomas. Because these lesions arise only with time, nonviral factors appear to be involved. We have karyotyped several primary tumors as well as a series of low-passage cell lines derived both from fibromatoses and from fibrosarcomas. The fibrosarcomas, but not the preneoplastic fibromatoses, show consistent abnormalities of one or both of two chromosomes, chromosome 8 (trisomy or duplication) and chromosome 14 (monosomy or translocation). The chromosomal abnormalities are not a direct consequence of the viral integration, which we have mapped to chromosome 15 by in situ hybridization. These results suggest that transgenic mice can be used to study the role(s) of cytogenetic changes in tumorigenesis and may direct the search for genes involved in tumor progression.

Animals↗
PubMed1989

E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

The E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) encodes positive- and negative-acting factors that regulate viral gene expression. The full-length ORF encodes a transactivator, and two transcriptional repressors are expressed from the 3' half of the ORF. Previous analysis has shown that a conserved C-terminal region of 101 amino acids, which is shared by E2 transactivator and repressor proteins, contains the specific DNA binding activity. Further analysis of the E2 transactivator shows that a conserved N-terminal domain of approximately 220 amino acids is crucial for the transcriptional activation function, whereas the variable internal region is not required. The E2 proteins bind to a sequence, ACCGN4CGGT, several copies of which are sufficient to constitute an E2-dependent enhancer. By using a gel retardation assay and proteins derived by in vitro transcription and translation, we were able to show that the E2 polypeptides bind as dimers to a single DNA binding site. The dimeric E2 proteins are stable in the absence of DNA and dimerization is mediated through the DNA binding domain. This may reveal an additional mechanism of repression that could potentially result from the formation of inactive heterodimers between transactivator and repressor species.

Amino Acid Sequence↗
PubMed1989

Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae. When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis. Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites. The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream. Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells. Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.

DNA-Binding Proteins↗
PubMed1989

The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes.

The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the E1, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human beta-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.

Cell Transformation, Viral↗
PubMed1989

Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1.

The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes three transcriptional regulatory proteins. The full-length open reading frame encodes a protein of 410 amino acids which functions as a transcriptional transactivator. Two transcriptional repressor proteins, E2-TR and E8/E2, contain the C-terminal 249 and 204 amino acids, respectively. We have expressed both the full-length E2 protein and the E2-TR repressor protein in insect cells, by using recombinant baculoviruses, and in mammalian COS-1 cells, by using a chimeric simian virus 40/BPV-1 virus. Analysis of the E2 proteins revealed that both the transactivator and repressor forms are phosphorylated predominately on serine residues at similar sites in both expression systems. By a combination of peptide mapping and site-directed mutagenesis techniques, the serine residues at positions 298 and 301 were determined to be the major phosphorylation sites of the BPV-1 E2 proteins.

Amino Acid Sequence↗
PubMed1989

Genetic assignment of multiple E2 gene products in bovine papillomavirus-transformed cells.

The E2 open reading frame of bovine papillomavirus type 1 has been shown genetically to encode at least three transcriptional regulatory factors, and three E2 specific proteins have been recently identified in virally transformed rodent cells. In this study, the genes encoding these E2 specific proteins have been determined. The 48-kilodalton (kDa) protein was identified as the product of a full-length E2 open reading frame cDNA, which confirmed that this polypeptide is the E2 transactivator. The 31-kDa E2 protein species, which is the most abundant E2 specific polypeptide, was identified by analysis of both bovine papillomavirus type 1 mutants and cDNAs to be the previously identified E2 transcriptional repressor, E2-TR, which results from translation initiation at an internal E2 ATG codon. The smallest E2 protein species, the 28-kDa polypeptide, was identified as the product of the E8/E2 fusion gene which results from translation of a spliced mRNA species.

Animals↗
PubMed1989

Cell-heritable stages of tumor progression in transgenic mice harboring the bovine papillomavirus type 1 genome.

Tumorigenesis of dermal fibroblasts in a line of transgenic mice carrying the BPV-1 genome was found to involve distinct proliferative stages. Cell cultures derived from normal skin, from benign proliferative fibromatoses, and from malignant fibrosarcomas each evidenced distinguishable, cell-heritable characteristics. The latent viral genome was transcriptionally inactive in normal-appearing skin and was activated in the dermal fibromatoses. Fibrosarcoma cells grew continuously in culture, formed domelike foci, and had a more rounded, anaplastic appearance. Independent cultures derived from the fibromatoses varied in their proliferative characteristics, which correlated well with the levels of viral gene expression. In contrast, progression to malignancy was not accompanied by a further increase in transgene activity, which strongly implicated cellular genetic changes in the later stages of tumorigenesis.

Animals↗
PubMed1989

Molecular mechanisms of transformation by the human papillomaviruses.

A variety of studies have now indicated that the papillomaviruses associated with cervical carcinoma, HPV-16 and HPV-18, contain two genes (E6 and E7) which have transforming properties. These genes are generally expressed in cervical carcinoma cells. The HPV-16 E7 protein has recently been shown to be a multi-functional protein possessing both transcriptional modulatory and cellular transforming properties similar to those described for adenovirus E1A proteins (1). E7 is able to transactivate the adenovirus E2 promoter and can cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The N-terminal 37 amino acids of all of the E7 proteins of the genital associated HPVs contain regions which are highly conserved and which are quite similar to portions of conserved domains 1 and 2 of adenovirus E1A. These domains in E1A are critical for cellular transformation properties and contain the amino acid sequences involved in binding the product of the retinoblastoma tumor suppressor gene (pRB). Results from a collaborative study with Ed Harlow and Nick Dyson (Cold Spring Harbor Laboratory) have shown that the E7 oncoprotein of HPV-16 can associate with the retinoblastoma gene product in vitro (2). The ability of the E7 proteins encoded by various HPVs to bind pRB has been examined using an in vitro complexing assay. E7 is not sufficient for transformation of human keratinocytes. The co-operation of the HPV-16 E6 and E7 genes has been shown to be important for transformation of these cells (3). Potential intracellular protein targets for E6 are being assessed.

Animals↗
PubMed1988

The human papillomaviruses. An overview.

Papillomaviruses are small DNA viruses that are associated with proliferative squamous epithelial lesions in many higher vertebrates. In humans, over 50 distinct human papilloma viruses (HPVs) have now been recognized, and each is associated with a specific set of clinical lesions. Approximately 15 of these viruses have been associated with benign proliferative lesions of the genital tract, and a subset of these has been regularly associated with vulvar, penile, and cervical lesions that are generally regarded as precancerous. The regular presence of these same HPV DNAs in cancers of the cervix, penis, and vulva has supported the notion that these HPVs may have an etiologic role in these malignancies. The biology and natural history of HPV infections are poorly understood. In addition to the clinically apparent lesions induced, HPV-DNA can often persist latently in tissues that appear entirely normal. The cellular, hormonal, immune, and other physiologic factors involved in determining whether or not a HPV infection will be clinically manifest are not known. In general, there is a paucity of information concerning the host cellular and humoral immune responses to a HPV infection, though clinical observations indicate that such responses probably play a major role in the resolution of HPV-associated diseases.

Cell Transformation, Viral↗
PubMed1988

The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A.

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Adenoviruses, Human↗
PubMed1988

The carboxy-terminal domain shared by the bovine papillomavirus E2 transactivator and repressor proteins contains a specific DNA binding activity.

The E2 open reading frame of bovine papilloma virus 1 (BPV-1) has been shown to encode both positive and negative acting transcriptional regulatory factors. The DNA binding properties of these factors were analysed to investigate the mechanism by which they might regulate viral gene expression. Polypeptides corresponding to the full-length E2 product and a shorter protein thought to represent the repressor function were synthesized in vitro by translation of T7 polymerase generated transcripts. Using rabbit antisera generated against synthetic peptides from the E2 open reading frame, it was possible to immunoprecipitate each of these products and show that each was capable of binding the same specific sequence located at several sites in the BPV-1 genome. This DNA binding property was mapped to a conserved carboxy-terminal domain of 101 amino acids by analysis of truncated polypeptides synthesized from the E2 open reading frame.

Bovine papillomavirus 1↗
PubMed1988

The bovine papillomavirus P2443 promoter is E2 trans-responsive: evidence for E2 autoregulation.

The bovine papillomavirus type 1 (BPV-1) P2443 promoter is located just upstream of the E2, E3, E4 and E5 open reading frames (ORFs) and is active in both transformed rodent cells and in productively infected warts. Analysis of viral RNA structures suggests that transcripts from this promoter encode the E2 transactivator as well as the E5 oncoprotein. To study expression of P2443, the chloramphenicol acetyltransferase (CAT) reporter gene was placed downstream of this promoter, deleting the E2 and E5 ORFs, in a plasmid which contained all BPV-1 upstream sequences including the long control region (LCR). By itself, this plasmid demonstrated a low level of activity in transient assays and could be transactivated to a high level by the full-length E2 product. Transactivation of P2443 expression by E2 required the LCR in cis in an orientation- and position-independent manner, suggesting that this transactivation was mediated through the E2 responsive enhancer elements (E2RE) located within the LCR. Primer extension analysis of the 5' ends of the viral transcripts from pooled cells expressing these P2443/CAT plasmids confirmed that E2 transactivation results in an increase in the steady-state levels of RNA initiated from the P2443 promoter. Furthermore, E2 transactivation of the P2443 promoter could be inhibited by the trans-repressor encoded by the 3' portion of the E2 ORF. Thus, expression of the E2 transactivator and the E5 oncoprotein is directly regulated by transcriptional factors encoded by the E2 ORF.

Animals↗
PubMed1988

Bovine papillomavirus type 1 E1 replication-defective mutants are altered in their transcriptional regulation.

Bovine papillomavirus type 1 (BPV-1) is capable of replicating as a stable, high-copy-number plasmid in transformed rodent cells. The BPV-1 E1 open reading frame (ORF) encodes multiple functions involved in viral DNA replication. Mutations which disrupt the translational integrity of the E1 ORF disable the viral genome from replicating as a stable plasmid and result in the integration of the viral genome into the host chromosome generally at a low copy number. Despite the low copy number of the integrated genomes, BPV-1 E1 mutants transform rodent cells to anchorage independence very efficiently, at levels equal to or greater than that of wild-type (wt) BPV-1. Studies were performed to provide insight into why these low-copy-number, replication-defective mutants are capable of expressing an equal or greater transformation potential than wt BPV-1. Analysis of viral RNA revealed higher rates of transcription per viral genome in cells harboring E1 mutated BPV-1 DNA than in cells containing wt BPV-1 DNA. Furthermore, the levels of viral RNA mapping the P89 promoter were found to be 15- to 35-fold higher in cells transformed by E1 mutated DNAs compared with wt BPV-1 transformants. This promoter controls expression of the BPV-1 E6 transforming gene and is regulated by the viral E2 gene products. The studies presented in this report determined that the E1 mutants were perturbed in their E2 transcriptional regulation, suggesting a possible explanation for the observed P89 induction. Mutations throughout the E1 ORF, in either of the two regions previously identified as encoding distinct replication functions, were altered in viral transcription.

Animals↗
PubMed1988

Evidence for cooperativity between E2 binding sites in E2 trans-regulation of bovine papillomavirus type 1.

The long control region of bovine papillomavirus type 1 (BPV-1) can function in an orientation- and position-independent manner as an E2-dependent enhancer. Dissection of the long control region has revealed two E2-responsive elements, E2RE1 and E2RE2, which map, respectively, between nucleotides 7611 and 7806 and between nucleotides 7200 and 7386 of the BPV-1 genome. In this study, we have carried out a detailed analysis of E2RE1, which has previously been shown to be involved in the regulation of the BPV-1 promoters P89 and P7940. One characteristic of E2RE1 is the presence of a pair of ACCN6GGT motifs (E2 binding sites) at each end of the element. To determine the contribution of these sites, as well as other sequences within E2RE1, to enhancer function, specific mutations and deletions were generated by oligonucleotide reconstruction. The functional analysis of these mutations confirmed that a pair of E2 binding sites was essential for E2-dependent enhancer activity but also indicated that cooperativity between the motifs at each end of E2RE1 creates a highly responsive element. Isolated ACCN6GGT motif pairs could also act as E2-dependent enhancers but at a significantly reduced level in comparison to the intact element. The sequences between the E2 binding sites in E2RE1 were not required for enhancer function and could actually block the enhancer activity of an isolated pair of E2 binding sites when positioned between the binding sites and the enhancer-deleted simian virus 40 early promoter.

Animals↗