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B Sugden

Publications and source records attributed to B Sugden.

At least 55 records · Page 3Linked to original sources
PubMed1991

Immortalizing genes of Epstein-Barr virus.

EBV immortalizes human B lymphocytes efficiently. Ten of its approximately 100 genes are expressed in these proliferating lymphoblasts and are candidates for mediating the changes central to the immortalization of the cell. Enough has been learned now about three of these viral genes to indicate that they are likely to be required for immortalization. As more is learned, additional genes of EBV will probably be found to support the process of immortalization of the host cell. EBNA-2 has been shown genetically to be required for EBV to immortalize an infected B lymphocyte. The biochemical activities of EBNA-2 that constitute this requirement have not been identified. Many experiments indicate that EBNA-2 affects the accumulation of specific viral and cellular RNAs. These effects, however, can be detected only in certain EBV-negative B-lymphoblastoid cells. It is, therefore, not clear that the known effects of EBNA-2 adequately explain its ubiquitous requirement in the immortalization of primary human B lymphocytes. LMP is likely to be required for immortalization because it can affect the growth properties of established human lymphoid and epithelial cells and can transform at least two established rodent cells to proliferate in an anchorage-independent manner. The structure of this viral protein, its position in the plasma membrane, many of its biochemical properties, as well as studies of its mutant derivatives are consistent with its acting as a growth factor receptor or affecting the activity of such a receptor. However, no biochemical activity has been assigned directly to LMP, and both its mechanism of action and its possible contribution to immortalization by EBV remain enigmatic. EBNA-1 presumably is required for EBV to immortalize a B lymphocyte because it is essential for the initiation of plasmid DNA replication by EBV. Circumstantial observations indicate also that EBNA-1 is probably necessary for sustaining viral DNA replication in the proliferating cell population. EBNA-1 may well affect the regulation of transcription of viral genes that themselves are required for immortalization. These roles of EBNA-1 are performed in part by its site-specific binding to the elements of oriP required in cis for the replication of EBV plasmid DNAs. It is probable that EBNA-1 also binds both to a set of cellular proteins that function in transcription and to a nonidentical set of cellular proteins that function in replication. EBV effects a fascinating phenotypic change in B lymphocytes it infects. It does so by using several viral genes that alter the physiology of the cell by different means.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) has a short half-life (V. R. Baichwal and B. Sugden, J. Virol, 61:866-875, 1987; K.P. Mann and D. Thorley-Lawson, J. Virol, 61:2100-2108, 1987), is localized in patches in the membrane (D. Liebowitz, D. Wang, and E, Kieff, J. Virol, 58:233-237, 1986), and associates with the cytoskeleton in EBV-immortalized B lymphocytes (D. Liebowitz, R. Kopan, E. Fuchs, J. Sample, and E. Kieff, Mol. Cell. Biol. 7:2299-2308, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol. 61:2100-2108, 1987). Deletion mutants of LMP that are either positive or negative in the induction both of anchorage-independent growth of BALB/c 3T3 cells (V. R. Baichwal and B. Sugden, Oncogene 4:67-74, 1989) and of cytotoxicity in a variety of cells (W. Hammerschmidt, B. Sugden, and V. R. Baichwal, J. Virol. 63:2469-2475, 1989) have been studied to identify the biochemical properties of this protein that correlate with its effects on cell growth. Mutant LMP proteins that are metabolically stable, do not associate with the cytoskeleton, and exhibit a diffuse plasma membrane localization also do not induce anchorage-independent growth in rodent cells or cytotoxicity in B lymphoblastoid cells. In contrast, a mutant of LMP that is functionally identical to the wild-type protein has a half-life, membrane localization, and cytoskeletal association similar or identical to those of LMP. These results are consistent with the hypothesis that LMP's rapid turnover, association with the cytoskeleton, and patching in the membrane are required for it to affect cell growth.

Animals↗
PubMed1991

The latent membrane protein oncoprotein resembles growth factor receptors in the properties of its turnover.

The latent membrane protein (LMP) of Epstein-Barr virus functions as an oncogene in rodent cell lines (D. Wang, D. Liebowitz, and E. Kieff, Cell, 43: 831-840, 1985; V. R. Baichwal and B. Sugden, Oncogene, 2: 461-467, 1988) and, therefore, is likely to be essential for immortalization of human B-lymphocytes by Epstein-Barr virus. LMP has a short half-life in Epstein-Barr virus-infected B-lymphoblastoid cells (V. R. Baichwal and B. Sugden, J. Virol., 61: 866-875, 1987; K. P. Mann and D. Thorley-Lawson, J. Virol., 61: 2100-2108, 1987) and in LMP-transformed rodent cell lines (V. R. Baichwal and B. Sugden, Oncogene 2: 461-467, 1988). The hypothesis that the turnover of LMP functions to down-regulate LMP activity has been tested by determining whether the turnover of LMP resembles that of several receptors for growth factors and neurotransmitters. The rapid turnover of LMP in transformed BALB/c 3T3 cells is blocked by cycloheximide, which indicates that turnover requires ongoing protein synthesis. Greater than 90% of newly synthesized LMP is present at the cell surface within 20 min of synthesis, and the detectable protein remains at this location for up to 6 h. If cells are grown in the presence of cycloheximide such that turnover of LMP is inhibited, an internalized pool of LMP can be detected; this observation indicates that turnover of LMP is likely to be preceded by internalization and that, once internalized, LMP is rapidly degraded. Also, this result indicates that the degradation of LMP, as opposed to its internalization, requires ongoing protein synthesis. The turnover of LMP and its biological activity (as assayed by cytotoxicity) are not regulated by factor(s) present only in serum, because the half-life of LMP in cells maintained in serum-free medium does not differ from that in the same cells grown in 5% calf serum. The rapid turnover, the requirement of protein synthesis for turnover, and the internalization of LMP are consistent with the functioning of this protein as a (ligand-dependent or independent) cell surface receptor.

Animals↗
PubMed1990

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.

Antibodies, Viral↗
PubMed1990

DNA replication of herpesviruses during the lytic phase of their life-cycles.

We have two goals in this review: the first is to relate what has been learned about DNA replication from the study primarily of herpes simplex virus type 1 (HSV-1); the second is to note briefly facets of this virus's mode of DNA replication that might serve as points of intervention for novel chemotherapeutic approaches in order to deal with primary and recurrent herpesvirus infections in man and animals. For the first goal we shall both summarize what has been learned and attempt to identify directions that may be pursued in order to further our understanding of DNA replication by herpesviruses. For the second goal we shall propose two schemes for the screening for drugs that might interfere uniquely with the DNA replication of this family of viruses.

Animals↗
PubMed1989

Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes.

Epstein-Barr virus (EBV), a herpes virus, infects human B lymphocytes in vitro and efficiently immortalizes them. About 10 of the approximately 100 genes of EBV are expressed in recently immortalized B cells and although there is circumstantial evidence that at least three of these may contribute to the process of immortalization, there is no direct evidence that any particular gene is required. We have developed a genetic analysis of EBV that uses a transformation-defective strain of the virus as a helper virus in conjunction with DNA that contains all of the viral cis-acting elements required for replication, cleavage and packaging during the lytic phase of the viral life cycle. This DNA can include viral genes required for immortalization that complement the transformation-defective virus strain. The DNA can be amplified and packaged by the products of the helper virus and the packaged DNA is infectious. We have analysed two viral genes expressed in immortalized cells and find that the gene encoding EBV nuclear antigen-2 is required for immortalization, whereas the gene for the EBV nuclear antigen leader protein is not.

Antigens, Viral↗
PubMed1989

The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels.

A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Epstein-Barr virus. This gene encodes the latent membrane protein of Epstein-Barr virus. When the gene was expressed at high levels, it was toxic to all cell lines tested, which included six human B-lymphoid lines as well as BALB/3T3, 143/EBNA-1, and HEp-2 cells. The BNLF-1 gene was previously shown to induce anchorage-independent and tumorigenic growth in Rat-1 and BALB/3T3 cells. We demonstrate here that only those mutations in the BNLF-1 gene that score positively in the anchorage-independent growth assay were cytotoxic when expressed at high levels. It is therefore possible that the same activities of the latent membrane protein that are necessary to induce anchorage-independent growth of some rodent cell lines also confer toxicity to many cell lines when expressed at high levels.

Animals↗
PubMed1989

A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection.

A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P.J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.L. Yates, N. Warren, and B. Sugden, Nature [London] 313:812-815, 1985) and viral transcription.

Antigens, Viral↗
PubMed1989

The multiple membrane-spanning segments of the BNLF-1 oncogene from Epstein-Barr virus are required for transformation.

The BNLF-1 gene from Epstein-Barr virus (EBV) induces anchorage-independent and tumorigenic growth in rodent cell lines. The BNLF-1 protein (also termed LMP) is a membrane protein, and its predicted amino acid sequence indicates that the protein has six membrane-spanning segments in addition to a short amino-terminal (approximately 25 amino acids) and a long carboxyl-terminal (approximately 200 amino acids) cytoplasmic domain. To identify the regions of the protein that are essential for its transforming activity, we have constructed deletion mutants of the BNLF-1 gene and tested them for transforming activity. Surprisingly, the entire carboxyl-terminal cytoplasmic domain is dispensable for transforming activity, whereas the putative membrane-spanning segments are essential. These observations indicate that BNLF-1 has a novel function that is distinct from the functions associated with other membrane-associated viral transforming proteins. We speculate that BNLF-1 is a receptor for a growth-promoting agent, with its trans-membrane domain involved in ligand binding, and its amino-terminal domain or cytoplasmic loops involved in coupling BNLF-1 to effector molecules in the cell, a situation analogous to the rhodopsin group of receptors.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus.

We have identified a cis-acting element of Epstein-Barr virus (EBV) that mediates viral DNA replication during the lytic phase of this virus's life cycle. This lytic origin of DNA replication, termed oriLyt, is complex in structure in that it contains multiple regions that are required for replication and additional DNA sequences that increase replication. One of the required regions of oriLyt can be functionally substituted by a transcriptional enhancing element. DNA replication mediated by oriLyt depends on EBV DNA polymerase and yields a concatemeric molecule. A vector, which contains both oriP (the EBV plasmid origin of replication) and oriLyt, can be maintained as a plasmid in latently EBV-infected cells and can be amplified 100- to 1000-fold in cells in which the lytic phase of the viral life cycle is induced.

Cell Line↗
PubMed1988

Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus.

The BNLF-1 protein is the only non-nuclear Epstein-Barr virus (EBV) encoded protein that has been detected in B-lymphocytes immortalized by EBV. We demonstrate that the BNLF-1 gene induces anchorage-independent growth and tumorigenic transformation of the murine cell-line, Balb/3T3. This demonstration extends the earlier observation that the BNLF-1 gene can transform Rat-1 cells. In addition we find that the BNLF-1 protein is located in the particulate fraction of cells, is phosphorylated, and is turned over with a half-life of 2.0 to 3.5 h in the BNLF-1 transformed Balb/3T3 cells, just as it is in EBV-genome-positive B-cell-lines.

Animals↗
PubMed1988

Plasmid origin of replication of Epstein-Barr virus, oriP, does not limit replication in cis.

Two plasmids encoding resistance to hygromycin-B or to the analog of neomycin, G418, and containing either one or two copies of the plasmid origin of replication, oriP, of Epstein-Barr virus (EBV) were introduced into an EBV-positive B-lymphoblastoid cell line. Two clones of cells containing both plasmids were analyzed for the number of copies of each plasmid when the cells were propagated in the absence or in the presence of one or both selective agents. Under all conditions tested, the plasmid with two copies of oriP behaved in cells as did the plasmid with one copy of this plasmid origin of replication.

B-Lymphocytes↗
PubMed1987

Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus.

The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV. We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase. This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography. The antiserum was able to immunoprecipitate BNLF-1 from cell lysates. We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines. Pulse-chase analyses with [35S]methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h. Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands. We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells. The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells.

Animals↗