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D T Rowe

Publications and source records attributed to D T Rowe.

35 records · Page 2Linked to original sources
PubMed1989

The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle.

A spliced cDNA spanning the Epstein-Barr virus BZLF1 gene expresses the BZLF1 protein and is active in inducing the virus productive cycle. A deletion mutant which lacks the N-terminal half of the protein is inactive. Cotransfection experiments in EBV-negative B-lymphocyte cell lines demonstrated that the BZLF1 gene activates the promoter for the BSLF2 + BMLF1 gene in the absence of any other EBV gene product. These results confirmed that the spliced BZLF1 gene is the transactivating gene structure in BamHI-Z.

Amino Acid Sequence↗
PubMed1987

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.

Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.

Antibodies, Monoclonal↗
PubMed1987

Novel nuclear antigens recognized by human sera in lymphocytes latently infected by Epstein-Barr virus.

We have used three latently infected cell lines, X50-7, JC-5, and Raji, to identify two new nuclear antigen complexes by Western immunoblotting with human anti-EBNA (Epstein-Barr Virus Nuclear Antigen) sera. One antigen complex, termed EBNA III, is composed of a group of high molecular weight proteins between 130 and 160 kDa and the other antigen complex, termed EBNA IV, is a size-related group of polypeptides between 28 and 62 kDa. Both the EBNA III and EBNA IV groups of proteins display variation in size among the different strains of EBV. Cell fractionation of X50-7, JC-5, Raji, and C16, a cell clone of P3HR1, showed that both new antigen complexes were completely recovered from the nuclei of latently infected lymphocytes as were previously described EBNA I and II. Because these new antigens are only detected by anti-EBNA sera in EBV infected cells, it seems likely that they may be encoded by the viral genome and play some role in the immortalization of lymphocytes by the virus.

Antigens, Viral↗
PubMed1987

Mapping of the gene coding for Epstein-Barr virus-determined nuclear antigen EBNA3 and its transient overexpression in a human cell line by using an adenovirus expression vector.

The open reading frame which lies within the Epstein-Barr virus (EBV) T2 cDNA isolated by Bodescot et al. (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:2611-2620, 1986) was inserted into a eucaryotic expression vector containing a strong adenovirus promoter. The T2 cDNA contains viral genomic sequences from the short BLRF3 open reading frame fused to the adjacent BERF1 long open reading frame. After transfection of human cells, the recombinant plasmid directed the expression of a 140-kilodalton protein. The expressed protein had the same molecular weight, subcellular localization, and immunological characteristics as the EBV-determined nuclear antigen EBNA3, which is made in lymphocytes latently infected with EBV. Immunoprecipitation of extracts of transfected cells labeled with [32P]phosphoric acid showed that the EBNA3 protein is phosphorylated.

Adenoviruses, Human↗
PubMed1986

Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt's lymphoma cells.

Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.

Antigens, Surface↗
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↗
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↗
PubMed1983

Mapping of a 14,000-dalton antigen to early region 4 of the human adenovirus 5 genome.

An early 14,000-dalton (14K) antigen of adenovirus 5, hitherto designated 10.5K and thought to be from early region 1 (E1), has been shown to be a product of region E4 on the following evidence. In KB cells infected with the adenovirus 5 mutants dl312 and dl313, containing large deletions in region E1, this antigen was produced in a form having the same mobility as that in wild-type infections. In a range of rodent cells transformed by adenovirus 5 DNA, the synthesis of 14K antigen and the ability of these cells to elicit an immune response to this protein both correlated with the presence of sequences from region E4 of the viral genome. A 14K polypeptide was synthesized in a cell-free system programmed with infected-cell mRNA and was found to be identical to the in vivo antigen in antigenicity, in electrophoretic mobility, and in [35S]methionine-containing tryptic peptides. After labeling with [35S]methionine and several 3H-amino acids, this in vitro product gave an N-terminal sequence identical to that expected from one of the open reading frames (reading region 3) in the DNA sequence for region E4 of Hérissé et al. (Nucleic Acids Res. 9:4023-4042, 1981). It is likely that this antigen is the same as the nucleus-associated 11K polypeptide from E4 described by other authors.

Adenoviruses, Human↗
PubMed1983

Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini.

Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins.

Adenoviruses, Human↗
PubMed1983

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants.

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic.

Adenoviruses, Human↗
PubMed1981

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.

Adenoviruses, Human↗