Viral DNA contamination is responsible for Epstein–Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein–Barr virus B-lymphoblastoid cell line

ABSTRACT Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.

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Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.80.7.1987 ◽

1983 ◽

Vol 80 (7) ◽

pp. 1987-1991 ◽

Cited By ~ 72

Author(s):

A. Henderson ◽

S. Ripley ◽

M. Heller ◽

E. Kieff

Keyword(s):

Tumor Cell ◽

Cell Line ◽

Epstein Barr Virus ◽

Tumor Cell Line ◽

Barr Virus ◽

Epstein Barr

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Development of suppressor T lymphocytes for Epstein-Barr virus-induced B-lymphocyte outgrowth during acute infectious mononucleosis: assessment by two quantitative systems

Blood ◽

10.1182/blood.v57.3.510.510 ◽

1981 ◽

Vol 57 (3) ◽

pp. 510-517 ◽

Cited By ~ 20

Author(s):

RT Schooley ◽

BF Haynes ◽

J Grouse ◽

C Payling-Wright ◽

AS Fauci ◽

...

Keyword(s):

T Lymphocytes ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

B Lymphocyte ◽

Acute Stage ◽

Cell Mediated Immunity ◽

Barr Virus ◽

Epstein Barr

Abstract A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

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The Clinical Use of Cord Blood-Derived Virus-Specific Cytotoxic T Lymphocytes Reactive Against Cytomegalovirus (CMV), Adenovirus, and Epstein-Barr Virus (EBV)

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2010.12.024 ◽

2011 ◽

Vol 17 (2) ◽

pp. S158

Author(s):

P.J. Hanley ◽

C. Martinez ◽

K. Leung ◽

C.R.Y. Cruz ◽

B. Savoldo ◽

...

Keyword(s):

T Lymphocytes ◽

Cord Blood ◽

Cytotoxic T Lymphocytes ◽

Epstein Barr Virus ◽

Clinical Use ◽

Barr Virus ◽

Epstein Barr

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Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

Journal of Virology ◽

10.1128/jvi.01518-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1195-1208 ◽

Cited By ~ 23

Author(s):

Chih-Chung Lu ◽

Ho-Ting Huang ◽

Jiin-Tarng Wang ◽

Geir Slupphaug ◽

Tsai-Kun Li ◽

...

Keyword(s):

Dna Replication ◽

Epstein Barr Virus ◽

Efficient Production ◽

Transcription Activator ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr ◽

Lytic Dna Replication

ABSTRACT Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

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