Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients

ABSTRACT LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

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Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2533 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2533-2542 ◽

Cited By ~ 173

Author(s):

S Lupton ◽

A J Levine

Keyword(s):

Dna Sequence ◽

Hela Cells ◽

Epstein Barr Virus ◽

Multicopy Plasmid ◽

Terminal Portion ◽

Plasmid Maintenance ◽

Cis Acting ◽

Barr Virus ◽

Epstein Barr ◽

Carboxy Terminal

The Epstein-Barr virus (EBV) genome becomes established as a multicopy plasmid in the nucleus of infected B lymphocytes. A cis-acting DNA sequence previously described within the BamHI-C fragment of the EBV genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984) allows stable extrachromosomal plasmid maintenance in latently infected cells, but not in EBV-negative cells. In agreement with the findings of Yates et al., deletion analysis permitted the assignment of this function to a 2,208-base-pair region (nucleotides 7315 to 9517 of the B95-8 strain of EBV) of the BamHI-C fragment that contained a striking repetitive sequence and an extended region of dyad symmetry. A recombinant vector, p410+, was constructed which carried the BamHI-K fragment (nucleotides 107565 to 112625 of the B95-8 strain, encoding the EBV-associated nuclear antigen EBNA-1), the cis-acting sequence from the BamHI-C fragment, and a dominant selectable marker gene encoding G-418 resistance in animal cells. After being transfected into HeLa cells, this plasmid persisted extrachromosomally at a low copy number, with no detectable rearrangements or deletions. Two mutations in the BamHI-K-derived portion of p410+, a large in-frame deletion and a linker insertion frameshift mutation, both of which alter the carboxy-terminal portion of EBNA-1, destroyed the ability of the plasmid to persist extrachromosomally in HeLa cells. A small in-frame deletion and linker insertion mutation in the region encoding the carboxy-terminal portion of EBNA-1, which replaced 19 amino acid codons with 2, had no effect on the maintenance of p410+ in HeLa cells. These observations indicate that EBNA-1, in combination with a cis-acting sequence in the BamHI-C fragment, is in part responsible for extrachromosomal EBV-derived plasmid maintenance in HeLa cells. Two additional activities have been localized to the BamHI-C DNA fragment: (i) a DNA sequence that could functionally substitute for the simian virus 40 enhancer and promoter elements controlling the expression of G-418 resistance and (ii) a DNA sequence which, although not sufficient to allow extrachromosomal plasmid maintenance, enhanced the frequency of transformation to G-418 resistance in EBV-positive (but not EBV-negative) cells. These findings suggest that the BamHI-C fragment contains a lymphoid-specific or EBV-inducible promoter or enhancer element or both.

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Genome Diversity of Epstein-Barr Virus from Multiple Tumor Types and Normal Infection

Journal of Virology ◽

10.1128/jvi.03614-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5222-5237 ◽

Cited By ~ 122

Author(s):

Anne L. Palser ◽

Nicholas E. Grayson ◽

Robert E. White ◽

Craig Corton ◽

Samantha Correia ◽

...

Keyword(s):

Immune System ◽

T Cell ◽

Geographic Variation ◽

Epstein Barr Virus ◽

Sequence Variation ◽

Host Immune System ◽

Barr Virus ◽

The World ◽

Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection.IMPORTANCEMost people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

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The last seven transmembrane and carboxy-terminal cytoplasmic domains of Epstein-Barr virus latent membrane protein 2 (LMP2) are dispensable for lymphocyte infection and growth transformation in vitro.

Journal of Virology ◽

10.1128/jvi.67.4.2006-2013.1993 ◽

1993 ◽

Vol 67 (4) ◽

pp. 2006-2013 ◽

Cited By ~ 64

Author(s):

R Longnecker ◽

C L Miller ◽

X Q Miao ◽

B Tomkinson ◽

E Kieff

Keyword(s):

Membrane Protein ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Cytoplasmic Domains ◽

Barr Virus ◽

Epstein Barr ◽

Virus Latent Membrane Protein ◽

Carboxy Terminal ◽

Latent Membrane Protein 2

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Analysis of Epstein–Barr virus (EBV) nuclear antigen 1 subtypes in EBV-associated lymphomas from Brazil and the United Kingdom

Journal of General Virology ◽

10.1099/0022-1317-80-10-2741 ◽

1999 ◽

Vol 80 (10) ◽

pp. 2741-2745 ◽

Cited By ~ 16

Author(s):

Jane MacKenzie ◽

Diane Gray ◽

Roberto Pinto-Paes ◽

Luis F. M. Barrezueta ◽

Alison A. Armstrong ◽

...

Keyword(s):

United Kingdom ◽

Epstein Barr Virus ◽

Sequence Variation ◽

Geographical Location ◽

Nuclear Antigen ◽

Nucleotide Sequence Analysis ◽

Barr Virus ◽

The United Kingdom ◽

Geographical Regions ◽

Epstein Barr

EBNA-1 is the only viral protein consistently expressed in all cells latently infected by Epstein–Barr virus (EBV). There is a high frequency of sequence variation within functionally important domains of EBNA-1, with five subtypes identified. Individuals may be infected with multiple EBV strains (classified according to EBNA-1 subtype), but Burkitt’s lymphoma (BL) tumours carry a single subtype and exhibit some subtype preference. Subtype variation has also been related to geographical location. In the present study EBNA-1 polymorphisms were examined in a series of haematological malignancies from two distinct geographical regions, Brazil and the United Kingdom. Nucleotide sequence analysis of the carboxy-terminal region of EBNA-1 in 34 cases revealed six distinct sequences, some of which are novel. A new subtype, named V-Ala, was identified. EBNA-1 subtype in tumours differed markedly according to geographical location. In contrast to previous studies, we found evidence of EBNA-1 sequence variation within individual BL tumour samples.

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