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Biomedical subjects

D DiMaio

Publications and source records attributed to D DiMaio.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1988

Bovine papillomavirus E2 gene regulates expression of the viral E5 transforming gene.

We have performed transient-expression experiments with CV1 monkey kidney cells to investigate the role of the bovine papillomavirus type 1 (BPV1) E2 gene in the regulation of the E5 transforming gene. Direct analysis of the 7-kilodalton open reading frame (ORF) E5 protein and measurements of the expression of an E5-chloramphenicol acetyltransferase fusion protein indicate that the efficient expression of ORF E5 requires the full-length E2 gene, which can be supplied in trans. The viral long control region is required in cis for this response to ORF E2, and it acts in a position- and orientation-independent fashion characteristic of a transcriptional enhancer. Deletion analysis suggests that the P2443 promoter is required for efficient expression of the E5 gene. The E2 repressor activity encoded in the 3' end of the E2 gene inhibits the expression of ORF E5. These effects define a major BPV1 regulatory circuit and appear to explain the transformation behavior of a variety of BPV1 mutants.

Acetyltransferases↗
PubMed1988

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.

Amino Acid Sequence↗
PubMed1987

Stimulation of cellular DNA synthesis by wild type and mutant bovine papillomavirus DNA.

Microinjection of recombinant plasmids containing bovine papillomavirus type 1 DNA into the nuclei of mouse C127 cells results in the stimulation of cellular DNA synthesis. Mutations in the viral E2 gene have no apparent effect on this activity even though the same mutations prevent efficient C127 cell focus formation and inhibit transactivation by this gene.

Animals↗
PubMed1987

Genetic and biochemical definition of the bovine papillomavirus E5 transforming protein.

Mutations surrounding the first methionine codon of the E5 transforming gene of bovine papillomavirus (type 1) were analyzed for their effect on cellular transformation and on the synthesis of the 7-kd E5 polypeptide. Frameshift mutations upstream of this methionine codon (bp 3879) affect neither transforming activity nor the ability to synthesize full-size E5 protein. In contrast, frameshift mutations distal to this position result in the inhibition of cell transformation and prevent synthesis or accumulation of E5 protein in cells containing the mutant viral genomes. Several in-frame mutations distal to the first methionine codon have a minimal effect on transforming activity but alter the electrophoretic mobility of the E5 protein in a manner consistent with the generated genetic alteration (deletion, insertion or substitution). In all cases where the protein is detected, it fractionates with cellular membranes and forms dimers. These studies indicate that (i) the methionine codon at bp 3879 serves as the initiation codon for the mature E5 protein, (ii) changing the charge of the E5 amino-terminus (from neutral to positive) does not prevent the association of this hydrophobic polypeptide with cellular membranes, and (iii) E5 amino-terminal mutations do not interfere with the ability of this polypeptide to form homodimers. We conclude that the major focus-inducing activity of the intact BPV genome is due to the function of the small polypeptide encoded in the 3' half of the E5 ORF.

Amino Acid Sequence↗
PubMed1987

Mutational analysis of open reading frame E4 of bovine papillomavirus type 1.

Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.

Animals↗
PubMed1986

Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity.

A series of mutations in open reading frame (ORF) E5 of bovine papillomavirus type 1 has been constructed to determine whether this putative gene is required for in vitro oncogenic transformation by viral DNA. Frameshift mutations at either of two different positions located exclusively in ORF E5 cause a substantial reduction in the ability of the cloned viral DNA to induce the appearance of transformed foci of mouse C127 cells. A genetic mapping experiment with one of the mutants indicates that this characteristic transformation defect is actually due to the constructed mutation in ORF E5. Analysis of 10 different mutants with sequence changes at a single position in the ORF showed that there is an exact correspondence between transformation-competence and the ability of the 3' half of ORF E5 to be correctly translated. The transformation defect of an ORF E5 frameshift mutant can be suppressed by a second mutation that restores the correct reading frame to most of the ORF, but not by one that restores the reading frame near the 3' end of the ORF. These results constitute strong genetic evidence that translation of ORF E5 is required for efficient transformation of mouse C127 cells by bovine papillomavirus DNA. Wild-type ORF E5 has the potential to encode a short hydrophobic protein or polypeptide domain.

Amino Acid Sequence↗