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D DiMaio

Publications and source records attributed to D DiMaio.

At least 19 recordsLinked to original sources
PubMed1992

Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells.

The 44-amino acid E5 transforming protein of bovine papillomavirus is the shortest protein known to induce tumorigenic transformation of fibroblasts. We showed previously that expression of the E5 protein activates the cellular beta receptor for platelet-derived growth factor (PDGF) and proposed that the activated receptor transmits the transforming signal to the cell. Here we use coimmunoprecipitation analysis to show that the E5 protein and the activated PDGF receptor exist in a stable complex in transformed mouse C127 cells. These results suggest a distinct mechanism of growth factor receptor activation and provide further evidence that the PDGF receptor is an important target of the E5 protein.

Animals↗
PubMed1992

Localization of bovine papillomavirus type 1 E5 protein to transformed basal keratinocytes and permissive differentiated cells in fibropapilloma tissue.

We examined expression of the E5 transforming protein of bovine papillomavirus type 1 (BPV-1) in naturally and experimentally infected bovine cells. Bovine conjunctival fibroblasts transformed in vitro by experimental infection with purified BPV-1 virions expressed significantly higher amounts of the 7-kDa E5 protein than BPV-1-transformed murine C127 cells. Indirect immunofluourescence analysis revealed a cytoplasmic, predominantly juxtanuclear, localization of E5 protein in the in vitro virus-transformed bovine cells. In naturally infected bovine skin fibropapilloma tissue, two widely separated sites of E5 protein synthesis were identified within the epithelial layers. Transformed basal layer keratinocytes throughout the tumor tissue expressed cytoplasmic E5 protein at a low uniform level. In addition, abundant amounts of cytoplasmic E5 protein with a granular staining pattern were detected in highly differentiated keratinocytes in close association with sites of viral capsid protein synthesis. These observations imply roles for the viral E5 oncogene in the growth transformation of basal epidermal keratinocytes as well as in the differentiation-linked process of viral maturation. Detection of a papillomavirus protein in the basal cell population of warts lends support to the hypothesis that these cells are maintained in a transformed state by continuous expression of a viral transforming gene.

Amino Acid Sequence↗
PubMed1992

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.

Amino Acid Sequence↗
PubMed1992

The putative E5 open reading frame of cottontail rabbit papillomavirus is dispensable for papilloma formation in domestic rabbits.

In the cottontail rabbit papillomavirus (CRPV)-rabbit system, recombinant CRPV DNA can induce papillomas. This investigation was undertaken to evaluate whether the E5 open reading frame (ORF) of CRPV is required for papilloma formation. The CRPV genome we utilized, CRPV-WA, was sequenced in the E5 region and was found to contain one deletion, two insertions, and one transition mutation compared with CRPV-KS, the CRPV genome that has been fully sequenced. Despite these differences, an intact E5 ORF is preserved, supporting the notion that this gene may serve a biological function. One frameshift and two in-frame mutations were constructed in the small region of the 5' end of the E5 ORF that follows the E2 stop codon and precedes the L2 ORF. Several hundred rabbit skin sites were inoculated with each DNA preparation with a jet injector to test the ability of three CRPV E5 mutant DNAs to induce papillomas. In vivo results showed that each of the mutants induced papillomas, and biochemical analysis demonstrated that the E5 mutations present in DNA inocula were retained in the papillomas. The frequency of papilloma formation, however, was generally lower with each of the CRPV E5 mutants than with wild-type CRPV DNA, particularly so for the E5 frameshift mutant, suggesting that although the recognized E5 ORF is not required in domestic rabbits for the induction of papillomas by CRPV DNA, it may facilitate their formation.

Amino Acid Sequence↗
PubMed1991

Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein.

The bovine papillomavirus E5 gene encodes a 44 amino acid membrane-associated protein that can induce tumorigenic transformation of rodent fibroblast cell lines. Genetic studies suggest that the E5 protein may transform cells by influencing the activity of cellular proteins involved in growth regulation. We report here that the endogenous cellular beta type receptor for the platelet-derived growth factor (PDGF) is constitutively activated in C127 and FR3T3 cells stably transformed by the E5 protein, but not in these cell types transformed by a variety of other oncogenes. In C127 cells, a metabolic precursor as well as the mature form of the receptor is activated by E5 transformation. Activation of the receptor also occurs upon acute E5-mediated transformation of these cells and precedes mitogenic stimulation in this system. Moreover, activation of the receptor by addition of PDGF or the v-sis gene to untransformed cells is sufficient to induce DNA synthesis and stable growth transformation. We propose that the PDGF receptor is an important cellular intermediate in the transforming activity of the bovine papillomavirus E5 protein. There is a short region of sequence similarity between the fibropapillomavirus E5 proteins and PDGF, suggesting that the E5 proteins may activate the PDGF receptor by binding directly to it.

Animals↗
PubMed1991

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.

3T3 Cells↗
PubMed1991

Biological properties of the deer papillomavirus E5 gene in mouse C127 cells: growth transformation, induction of DNA synthesis, and activation of the platelet-derived growth factor receptor.

We determined the biological activities of the 44-amino-acid deer papillomavirus (DPV) E5 protein in mouse C127 cells. The DPV E5 gene can induce focus formation, stable and acute morphologic transformation, and DNA synthesis, and it activates the platelet-derived growth factor (PDGF) beta receptor as assessed by increased constitutive tyrosine phosphorylation of mature and precursor receptor forms. Thus, the DPV E5 protein has biological activities similar to those of the closely related E5 protein from bovine papillomavirus type 1. Moreover, like the bovine papillomavirus type 1 E5 protein, the DPV E5 protein shares a short region of sequence similarity with the B chain of PDGF. These results show that activation of the PDGF receptor is a general property of fibropapillomavirus E5 proteins, lending support to our previous proposal (L. Petti, L. Nilson, and D. DiMaio, EMBO J. 10:845-855, 1991) that activation of the PDGF receptor may play a central role in transformation of fibroblasts by E5 proteins.

Amino Acid Sequence↗
PubMed1990

Integrated HPV 1 genomes in a human keratinocyte cell line can be transactivated by a SV40/BPV1 recombinant virus which expresses BPV1 E2 proteins.

This paper describes studies carried out on an HPV 1 carrying human keratinocyte cell line (SVD2) and two subclones of it. Although these lines contain multiple copies of HPV 1 genomes, in situ hybridization revealed that integration was restricted to band q33 on the long arms of chromosome 2. An E4 1.25-kb mRNA was specifically identified by Northern blotting and a PCR generated cDNA confirmed the presence of the E1/E4 spliced mRNA which is abundant in HPV 1 containing papillomas. Infection of an SVD2 subclone (SVD2 (cyst) sp) with a SV40/BPV1 recombinant virus which expresses the BPV1 E2 48-kb transactivator protein enhanced HPV 1 transcription at least 20-fold.

Cell Line, Transformed↗
PubMed1990

Bovine papillomavirus E2 repressor mutant displays a high-copy-number phenotype and enhanced transforming activity.

The methionine codon at bovine papillomavirus type 1 nucleotide 3091 was mutated to determine whether it may serve as an initiation codon for an E2 transcriptional repressor protein and to determine the role of the repressor in the biological activities of the virus. A series of transient expression experiments with CV1 cells documented that the mutation reduced expression of repressor activity from the viral genome and resulted in increased expression of the E5 transforming gene. Viral genomes containing the mutation displayed enhanced transforming activity in several assays in mouse C127 cells, including focus formation, colony formation in agarose, and tumorigenicity. In transformed cells, the mutant viral DNA was maintained as a plasmid with approximately 500 genomes per cell, whereas the wild-type copy number was approximately 75. These results indicate that the wild-type bovine papillomavirus type 1 genome encodes an E2 repressor protein that moderates the viral transforming activity and allows maintenance of the viral DNA at a relatively low copy number.

Animals↗
PubMed1989

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.

A series of mutations in open reading frames (ORFs) E6 and E7 of bovine papillomavirus type 1 (BPV1) was constructed to analyze the roles of these ORFs in transformation of mouse C127 cells. The mutations were designed to prevent synthesis of specific proteins encoded by these genes. None of the mutations caused a decrease in the focus-forming activity of the full-length viral genome or in the ability of the viral DNA to replicate as a high-copy-number plasmid. Analysis of these mutants in the absence of a functional BPV1 E5 gene revealed a weak focus-forming activity encoded by ORF E6. Mutations preventing synthesis of the E6 protein did cause defects in anchorage-independent growth and tumorigenicity of transfected and transformed cells. However, a frameshift mutation between the first and second ATG codons of ORF E6 did not inhibit induction of colony formation, suggesting that translation from the first methionine codon is not required. Mutations that inactivated ORF E7 or E6/E7 individually did not inhibit induction of colony formation in agarose. However, a defect in this activity was caused by simultaneous disruption of both ORF E7 and ORF E6/E7 when they were expressed from the full-length viral genome but not when they were expressed under the control of a retrovirus long terminal repeat. These results suggest that translation of both ORF E6 and the 3' end of ORF E7 is required for efficient induction of anchorage-independent growth by the intact BPV1 genome.

Animals↗
PubMed1989

Transforming activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the smallest transforming protein yet described. Previous results from our laboratory indicate that a hydrophobic core and specific carboxyl-terminal amino acids are required for the E5 protein to exert its transforming function. In this study, additional substitution mutations were generated in the E5 gene to determine the minimal amino acid sequence requirements for focus formation in mouse C127 cells. In most cases examined, substitution of the hydrophobic middle third of the E5 protein with unrelated hydrophobic sequences severely inhibited transforming activity. However, we have identified one hydrophobic amino acid sequence apparently unrelated to the wild-type one that can replace the middle third of the wild-type E5 protein without affecting the ability of the protein to stably transform cells or interact with cell membranes. Furthermore, a mutant E5 protein in which only the carboxyl-terminal 16 amino acids of the protein have been derived from E5 sequences retains transforming activity. Since several residues in the carboxyl-terminal portion of the E5 protein can be freely substituted with different amino acids (B. H. Horwitz, A. L. Burkhardt, R. Schlegel, and D. DiMaio, Mol. Cell. Biol. 8:4071-4078, 1988), the results reported here imply that much of the specific information necessary for cell transformation can be supplied by a subset of the carboxyl-terminal 16 amino acids of this protein.

Amino Acid Sequence↗
PubMed1989

Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein.

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.

Amino Acid Sequence↗
PubMed1989

Bovine papillomavirus type I induces resistance to Ca+(+)-induced terminal differentiation in murine keratinocytes.

Bovine papillomavirus type 1 (BPV-1) was expressed in established mouse BALB/MK epidermal keratinocytes. In each of the transfected cell lines, DNA synthesis was stimulated by epidermal growth factor (EGF) and inhibited by type beta transforming growth factor to an extent similar to that in parental cells. In contrast, the BPV-1-transfectants were resistant to the induction of terminal differentiation by extracellular Ca(+)+. First, BPV transfectants continued to respond to EGF in the presence of Ca(+)+, whereas the growth of BALB/MK cells was arrested. The characteristics of EGF-binding were identical in the two cell lines. Second, the Ca+(+)-switch failed to induce the activation of epidermal transglutaminase in BPV-1-transfectants. Thus, BPV-1 caused mouse keratinocytes to become resistant to Ca+(+)-induced differentiation without otherwise affecting the control of cell growth.

Animals↗
PubMed1988

Bovine papillomavirus mutant temperature sensitive for transformation, replication and transactivation.

The genetic analysis of the papillomaviruses has been hampered by the lack of mutants conditionally defective for viral biological activities. We report here the construction and characterization of a temperature-sensitive papillomavirus mutant. The mutation is predicted to insert the sequence Pro-Arg-Ser-Arg into the N-terminal half of the bovine papillomavirus type 1 (BPV1) ORF E2 protein, the major viral regulatory protein. The cloned mutant viral DNA displays temperature-sensitive defects in the induction of focus formation in mouse C127 cells, in its establishment as an extrachromosomal plasmid and in transactivation of a BPV1 enhancer. Genetic experiments confirm that this pleiotropic phenotype is caused by the insertion mutation in ORF E2 and that the transformation and replication defects of the mutant at 37 degrees C are corrected in trans by wild-type E2 gene activity. Most cell lines stably transformed by the mutant at 32.5 degrees C display a reduced ability to overgrow a monolayer of normal cells following temperature shift to 37 degrees C and the mutant viral DNA after temperature shift is present in decreased copy number and/or in an integrated state. These results provide strong genetic evidence that continued ORF E2 activity is required for maintenance of BPV1-induced transformation and for normal viral DNA replication.

Bovine papillomavirus 1↗
PubMed1988

Efficient transactivation and morphologic transformation by bovine papillomavirus genes expressed from a bovine papillomavirus/simian virus 40 recombinant virus.

To efficiently introduce bovine papillomavirus type 1 genes into cultured cells, we constructed a hybrid viral genome in which the simian virus 40 early region is replaced with a segment of the bovine papillomavirus type 1 transforming region. High-titer stocks of simian virus 40 virions containing the recombinant genome were produced in monkey cells that express simian virus 40 large tumor antigen. Cells infected with this virus efficiently expressed the bovine papillomavirus type 1 E2 and E5 genes. Expression of the E2 gene caused transactivation of genes linked to the bovine papillomavirus type 1 control region, resulting in up to a 1000-fold induction. At high multiplicity of infection of a cell line containing an integrated reporter gene, most cells were infected and responded to transactivation. Within 48 hr of infection with wild-type virus but not with an open reading frame E5 mutant, mouse C127 cells displayed dramatic changes in morphology and growth characteristics similar to those seen in tumorigenic transformation. This system can be used to determine the acute cellular response to introduction of bovine papillomavirus type 1 transforming and regulatory genes; it can also be used to induce foreign genes stably incorporated into cultured mammalian cells.

Bovine papillomavirus 1↗