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P E Branton

Publications and source records attributed to P E Branton.

At least 73 records · Page 4Linked to original sources
PubMed1988

Identification of the phosphorylation sites in early region 1A proteins of adenovirus type 5 by amino acid sequencing of peptide fragments.

We have mapped the positions of three of the phosphorylation sites on the 289 and 243 residue (289R and 243R) early region 1A (E1A) proteins of human adenovirus type 5 (Ad5). These proteins, which play roles in both transcriptional control and oncogenic transformation, have identical sequences except for the presence in 289R of 46 additional internal amino acids. Phosphorylation was detected exclusively at serine residues. E1A proteins purified from [35S]methionine- or [32P]orthophosphate-labeled Ad5-infected cells were digested with trypsin, and two phosphopeptides were isolated by reverse-phase chromatography and subjected to automated Edman degradation. The major species was shown to contain a single phosphorylation site at Ser-219. The second phosphopeptide was shown to contain at least one phosphorylation site at Ser-231. A third phosphorylated tryptic peptide could not be eluted from the column but was isolated using an E1A-specific rat monoclonal antibody. Following subcleavage by Staphylococcus aureus V-8 protease, this peptide was shown to contain at least one phosphorylation site at Ser-89. The present data indicate that both the 289R and 243R E1A proteins are phosphorylated at the same sites, at least one in the amino terminal half of the molecule, and at least two toward the carboxyl terminus.

Adenoviridae↗
PubMed1988

Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5.

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

Adenovirus Early Proteins↗
PubMed1987

Binding of cellular polypeptides to human adenovirus type 5 E1A proteins produced in Escherichia coli.

Cellular proteins of 300, 107, 105, 68 and 65 kDa have previously been shown to associate specifically with the early region 1A (E1A) proteins of human adenovirus type 5. In the present study we report that, to varying degrees, these proteins also were capable of binding to E1A products produced in Escherichia coli from plasmids carrying cDNAs corresponding to the 1.1- and 0.9-kb E1A mRNAs. When these purified E1A proteins were mixed in solution with extracts from mock-infected human cells, the 68- and 65-kDa species bound very efficiently to the 1.1-kb mRNA product and somewhat less so with that of the 0.9-kb mRNA. The 107-, 105-, and 300-kDa species bound poorly, if at all, to both E1A products. Using the E1A 1.1-kb mRNA product which had been covalently attached to Sepharose beads, the 68-, 65-, and 300-kDa species bound efficiently, and binding of protein which migrated in SDS gels in the region of the 107- and 105-kDa species was also observed. In addition to these proteins, several other cellular polypeptides of 30, 33, 75, 95, 150, 180, and greater than 300 kDa were shown to bind to E1A-Sepharose and thus may also be E1A-binding proteins. The present data confirm the specificity of the previously identified cellular proteins for E1A products and show that binding of the 300-, 65-, and 68-kDa species does not require the presence of any other viral polypeptide. In contrast, the inefficient binding of the 107- and 105-kDa species to Escherichia coli-expressed E1A protein may suggest that these interactions require either eukaryotic-specific post-translational modifications of the E1A protein, or the presence of additional Ad5 gene products.

Adenovirus Early Proteins↗
PubMed1987

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.

Acylation↗
PubMed1985

Analysis of multiple forms of human adenovirus type 5 E1A polypeptides using an anti-peptide antiserum specific for the amino terminus.

Studies were carried out to further characterize the proteins coded for by the early 1A region (E1A) of human adenovirus type 5 (Ad5) in lytically infected cells using an antiserum prepared against a synthetic octapeptide corresponding to the amino termini of E1A products as well as another anti-peptide serum specific for the carboxy termini. Both sera precipitated the same collection of four major and two minor acidic phosphoproteins and it was confirmed that each of the 1.1- and 0.9-kb E1A mRNAs code for two major and one minor species. These data also indicated that none of the E1A species was produced by proteolytic cleavage. The deletion mutant dl313 which lacks DNA coding for the last 70 C-terminal amino acids of E1A products also produced multiple species which suggested that post-translational modifications involved in their generation do not take place in this region of the proteins. The N-terminal serum was effective in detecting neither the truncated 1.1-kb mRNA product predicted for the mutant hr1 nor the product of the small 0.6-kb E1A mRNA, suggesting that these species are either very short-lived in infected cells or exist in a conformation in which the amino terminus is inaccessable to the antibody.

Adenoviruses, Human↗
PubMed1985

Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1.

It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV-1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation.

Adenovirus Early Proteins↗
PubMed1985

Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides.

Antisera prepared against synthetic peptides corresponding to the amino and carboxy termini of human adenovirus type 5 (Ad5) early region 1A (E1A) proteins were used to identify polypeptides that are associated with these viral species in lytically infected KB cells. Proteins were sought which coprecipitated with E1A polypeptides using both sera and which were not recognized in extracts from mock-infected cells by either serum. Four such species were identified with apparent molecular weights of 68K, 65K, and a doublet at about 105K. A fifth species migrating with a molecular weight in excess of 250K was also identified consistently with E1A-C1 but not E1A-N1 serum. Addition of an excess of the appropriate synthetic peptide to the immunoprecipitation mixtures prevented the precipitation of all of these species. Mixing experiments demonstrated that all species were cellular proteins expressed in normal uninfected KB cells and in addition showed that an association with E1A proteins could take place in vitro. Studies carried out with the mutants pm975 and hr1 indicated that while the 105K doublet and the greater than 250K species were found with the products of both the 1.1- and 0.9-kb E1A mRNAs, 65K and 68K appeared to be primarily associated with those of the 1.1-kb mRNA. Finally, the 105K doublet and greater than 250K were shown to be phosphoproteins. These data indicated that Ad5 E1A proteins may function in a complex with cellular polypeptides which includes species of 105K, 68K, 65K, and possibly a large protein of greater than 250K.

Adenovirus Early Proteins↗
PubMed1985

Transformation by human adenoviruses.

When, approximately 10 years ago, it was shown that the functions essential for cell transformation were localized in a small region of the adenovirus genome, a DNA segment which at that time was thought to be capable of encoding two or three average-sized proteins at most, it seemed reasonable to hope that an understanding of the mechanisms by which adenoviruses transform cells might be quickly achieved. While such optimism might be forgiven, it was quite clearly naive in the extreme. As a consequence of mRNA splicing and the use of overlapping reading frames the number of proteins encoded within E1 is 2-3-times greater than would have been predicted a decade ago, and post-translational modifications may add another dimension of complexity. In fact it has taken nearly all of the past decade just to identify the proteins encoded in E1 and to characterize them in the most rudimentary way. However, we have now entered a period in which new information is accumulating at an extremely rapid rate as a result of several major technical and fundamental advances. Chief among these are the use of recombinant DNA techniques, particularly site-directed mutagenesis, which combined with methods for introducing mutations made in cloned sequences back into infectious virus, clearly represents a powerful approach to studying the functions of transforming proteins. In addition, the ability to express transforming proteins in bacteria and to produce large amounts of highly purified proteins which previously were only just detectable in infected and transformed cells is a major breakthrough. Advances in immunological techniques, particularly the development of monoclonal antibodies and antisera against synthetic peptides, have enormously simplified the task of detecting and characterizing E1 proteins. Finally, recent results suggesting that adenovirus transforming proteins may be functionally and structurally similar to other oncogenes brings a new perspective to the study of oncogenic transformation. Have all the proteins involved in transformation by adenoviruses been identified? It seems probable that all those virally coded proteins which play a major role are now known but of course minor players in the cast could still be waiting in the wings. We have pointed out that viral functions encoded outside region E1 may have some importance at least in initiation of transformation by virions and have speculated on the possibility that one or more of these may be involved in the integration of viral DNA into the host cell chromosome.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗
PubMed1985

Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells.

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces two mRNAs coding for phosphoproteins of 289 and 243 residues (289R and 243R). Each of these products has been shown to migrate on sodium dodecyl sulfate gels as two major and two minor species. In the present study, the stabilities of E1A polypeptides, as well as those of some other early Ad5 proteins, were studied in infected KB cells that were pulse-labeled with [35S]methionine and then chased in the presence or absence of cycloheximide. The E1B 58,000- and 19,000-molecular-weight proteins (58K and 19K proteins; 496R and 176R) as well as the E2A 72K DNA-binding protein were relatively stable over the 4-h chase period; turnover was less than 30%. The E1A species were considerably more unstable, with an overall half-life of about 60 min. Interestingly, it was found that when cycloheximide was present during the chase, E1A proteins were much more stable, and the half-life increased to about 240 min. Analysis of the stabilities of individual E1A species indicated that the products of the 1.1-kilobase mRNA (289R) had half lives (about 55 min) somewhat shorter than those (about 90 min) of the 0.9-kilobase mRNA products (243R). In addition, the faster-migrating species produced from each mRNA (molecular weights, 48,500 and 45,000) had significantly shorter half-lives than did the slower-migrating species (52,000 and 50,000). In the presence of cycloheximide, the faster-migrating species were still quite short-lived, but the half-lives of the 52K and 50K species were considerably increased. An examination of the kinetics of turnover of the various E1A species suggested that the faster-migrating forms may be precursors to the slower-migrating ones. Somewhat similar stabilities were also found for the various E1A species in Ad5-transformed 293 cells.

Adenoviruses, Human↗
PubMed1985

Protein kinase and ATP-binding activity associated with the 72-kdalton single-stranded DNA-binding protein from early region 2A of human adenovirus type 5.

We have developed monoclonal antibodies which react specifically with the human adenovirus type 5 early region 2A 72-kdalton phosphoprotein (72 kDa) and its 48-kdalton proteolytic cleavage product (48 kDa) and have used these antibodies to study a number of properties of these viral polyPeptides. Fluorescent antibody staining indicated that the 72 kDa was found almost entirely in the nucleus, generally in discreet patches. Preparations of the 72 kDa, purified by immunoprecipitation or by single-stranded DNA-cellulose column chromatography and incubated with [gamma-32P]ATP, were found to contain protein kinase activity. Using photoaffinity labelling with 8-azido-[alpha-32P]ATP, the 72 kDa was shown to be an ATP-binding protein. The ATP-binding site was probably in the amino terminal region because the 48 kDa which lacks approximately 120 residues at the amino terminus failed to bind ATP. Experiments carried out with immunoprecipitates from wild-type and temperature-sensitive (ts 125) infected cells suggested that the ts mutant-induced kinase activity was not more thermolabile than the wild-type protein. Thus although the present results indicated that protein kinase activity is associated with the 72 kDa, it is still unclear whether such activity is intrinsic to the 72 kDa or present in an enzyme associated with it. While it is probably not intrinsic to the carboxy terminal region affected by the ts 125 mutation, it is still possible that it resides in an amino terminal domain.

Adenosine Triphosphate↗
PubMed1984

The kinetics of synthesis of early viral proteins in KB cells infected with wild-type and transformation-defective host-range mutants of human adenovirus type 5.

We have studied the kinetics of early adenovirus type 5 (Ad5) protein synthesis during lytic infection of KB cells by wild-type (wt) and transformation-defective host-range (hr) mutants. Proteins encoded within four early regions were studied: early region 1A (E1A: 1.5 to 4.5 map units, mu), E1B (4.5 to 11.2 mu), E2A (61.6 to 74.9 mu), and E4 (91.4 to 99.1 mu). Synthesis of E1A products, the first to appear during wt lytic infection, was detectable within 2 h after injection, reached a peak within the next hour, then declined to very low levels by 7 h post-infection. Synthesis of E2 and E4 proteins began at about 3 h post-infection, was maximal by 6 h and thereafter declined sharply. The E1B 19K and 58K proteins were first detected around 3 h post-infection and, after reaching maximal levels of expression by 8 h, declined to lower levels by 12 h post-infection. Infections with the E1A mutant hr3 were characterized by greatly depressed levels of early expression of E1B, E2 and E4 polypeptides but protein synthesis from these regions appeared to recover at late times. The pattern of expression exhibited by the E1B mutant hr6 revealed delayed and reduced levels of expression of E1B, E2 and E4 protein synthesis but increased levels of E1A protein synthesis. These results are consistent with the reported role of E1A gene products in the activation of early gene expression and, in addition, suggest that a function encoded in E1B may also influence the expression of Ad5 early genes at early and late times.

Adenoviruses, Human↗
PubMed1984

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.

Adenoviridae↗
PubMed1984

Peptide maps and N-terminal sequences of polypeptides from early region 1A of human adenovirus 5.

Experiments exploring the reasons for a multiplicity of products from early region 1A of adenovirus 5 are described. Labeled early region 1A products from wild-type virus were synthesized in infected cells and in a cell-free system programmed with mRNA from infected cells, immunoprecipitated specifically with an antipeptide serum, E1A-C1, directed against the C-terminal sequence of E1A products, and separated by gel electrophoresis. Two-dimensional maps of [35S]methionine-labeled peptides were consistent with antigens of 52,000 daltons (52K) and 48.5K being from the 13S mRNA and antigens of 50K, 45K, and 35K from the 12S mRNA. Partial N-terminal sequences of 52K, 50K, 48.5K, and 45K synthesized in vitro showed that each of these antigens was initiated at the predicted ATG at nucleotide 560 in the DNA sequence. These results eliminate multiple initiation sites and proteolytic cleavage at the N-terminal end as sources of antigen diversity. Peptide maps and N-terminal sequences were obtained in a similar way for E1A products from the Ad5 deletion mutant dl1504, which lacks the normal initiator codon. As predicted, these polypeptides are initiated at the next ATG, 15 codons downstream in the wild-type sequence. These results are discussed in relation to Kozak's ribosomal scanning model.

Adenoviruses, Human↗
PubMed1984

Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.

Adenoviruses, Human↗
PubMed1984

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Animals↗
PubMed1983

Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells.

The intracellular localization of tumor antigens of human adenovirus type 5 (Ad5) during lytic infection of KB cells has been studied. The cells were pulse labeled with [35S]methionine early after infection and early proteins of 58,000 D (58K), 44K, 19K, 18.5K, and 14K detectable by immunoprecipitation with hamster antitumor serum were assayed for association with cytoplasm, nucleoplasm, chromatin, cytosol, cytoskeleton, and membranes. The 44,000 D (44K) tumor antigen encoded in early region 1A (E1A: 0-4.4%) was recovered in approximately equal amounts from cytoplasmic and nucleoplasmic fractions of pulse-labeled cells and within the cytoplasmic compartment was found in the cytosol as well as associated with the cytoskeleton. The E1B-58K (E1B: 4.5-11.2%) antigen was also found to be associated with the cytoplasmic and nucleoplasmic fractions in approximately equal amounts but unlike the E1A-44K showed no affinity for cytoskeletons. Pulse-chase and immunofluorescence experiments suggested the 58K antigen accumulated in the nucleus late in infection. The E1B-19K antigen was found almost exclusively associated with the membrane fraction of infected KB cells and was resolved in polyacrylamide gels into two related species of 18.5K and 19K. Immunofluorescence studies on the E1B 18.5-19K doublet suggested that within a population of infected HeLa cells a small minority seemed to be expressing copious amounts of stainable antigen. Cell fractionation and immunofluorescence studies showed that the E4-14K antigen was a nuclear protein and the only antigen in this study which showed a significant association with a nuclear subfraction composed almost entirely of histones. The implications of these findings for the roles of the Ad5 tumor antigens in lytic infection and transformation are discussed.

Adenoviruses, Human↗