Abstract
Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
Full Text
The Full Text of this article is available as a PDF (482.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Altiok E., Minarovits J., Hu L. F., Contreras-Brodin B., Klein G., Ernberg I. Host-cell-phenotype-dependent control of the BCR2/BWR1 promoter complex regulates the expression of Epstein-Barr virus nuclear antigens 2-6. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):905–909. doi: 10.1073/pnas.89.3.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birx D. L., Redfield R. R., Tosato G. Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related disorders. N Engl J Med. 1986 Apr 3;314(14):874–879. doi: 10.1056/NEJM198604033141403. [DOI] [PubMed] [Google Scholar]
- Bodescot M., Perricaudet M., Farrell P. J. A promoter for the highly spliced EBNA family of RNAs of Epstein-Barr virus. J Virol. 1987 Nov;61(11):3424–3430. doi: 10.1128/jvi.61.11.3424-3430.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Contreras-Brodin B. A., Anvret M., Imreh S., Altiok E., Klein G., Masucci M. G. B cell phenotype-dependent expression of the Epstein-Barr virus nuclear antigens EBNA-2 to EBNA-6: studies with somatic cell hybrids. J Gen Virol. 1991 Dec;72(Pt 12):3025–3033. doi: 10.1099/0022-1317-72-12-3025. [DOI] [PubMed] [Google Scholar]
- Contreras-Salazar B., Klein G., Masucci M. G. Host cell-dependent regulation of growth transformation-associated Epstein-Barr virus antigens in somatic cell hybrids. J Virol. 1989 Jun;63(6):2768–2772. doi: 10.1128/jvi.63.6.2768-2772.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dillner J., Kallin B. The Epstein-Barr virus proteins. Adv Cancer Res. 1988;50:95–158. doi: 10.1016/s0065-230x(08)60436-4. [DOI] [PubMed] [Google Scholar]
- Fåhraeus R., Fu H. L., Ernberg I., Finke J., Rowe M., Klein G., Falk K., Nilsson E., Yadav M., Busson P. Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int J Cancer. 1988 Sep 15;42(3):329–338. doi: 10.1002/ijc.2910420305. [DOI] [PubMed] [Google Scholar]
- Gavioli R., De Campos-Lima P. O., Kurilla M. G., Kieff E., Klein G., Masucci M. G. Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5862–5866. doi: 10.1073/pnas.89.13.5862. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gavioli R., Kurilla M. G., de Campos-Lima P. O., Wallace L. E., Dolcetti R., Murray R. J., Rickinson A. B., Masucci M. G. Multiple HLA A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4. J Virol. 1993 Mar;67(3):1572–1578. doi: 10.1128/jvi.67.3.1572-1578.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gratama J. W., Oosterveer M. A., Weimar W., Sintnicolaas K., Sizoo W., Bolhuis R. L., Ernberg I. Detection of multiple 'Ebnotypes' in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx. J Gen Virol. 1994 Jan;75(Pt 1):85–94. doi: 10.1099/0022-1317-75-1-85. [DOI] [PubMed] [Google Scholar]
- Gratama J. W., Oosterveer M. A., Zwaan F. E., Lepoutre J., Klein G., Ernberg I. Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implications for sites of viral latency. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8693–8696. doi: 10.1073/pnas.85.22.8693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gregory C. D., Rowe M., Rickinson A. B. Different Epstein-Barr virus-B cell interactions in phenotypically distinct clones of a Burkitt's lymphoma cell line. J Gen Virol. 1990 Jul;71(Pt 7):1481–1495. doi: 10.1099/0022-1317-71-7-1481. [DOI] [PubMed] [Google Scholar]
- Hennessy K., Fennewald S., Hummel M., Cole T., Kieff E. A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7207–7211. doi: 10.1073/pnas.81.22.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hennessy K., Kieff E. One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5665–5669. doi: 10.1073/pnas.80.18.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herbst H., Dallenbach F., Hummel M., Niedobitek G., Pileri S., Müller-Lantzsch N., Stein H. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4766–4770. doi: 10.1073/pnas.88.11.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones C. H., Hayward S. D., Rawlins D. R. Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites. J Virol. 1989 Jan;63(1):101–110. doi: 10.1128/jvi.63.1.101-110.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerr B. M., Lear A. L., Rowe M., Croom-Carter D., Young L. S., Rookes S. M., Gallimore P. H., Rickinson A. B. Three transcriptionally distinct forms of Epstein-Barr virus latency in somatic cell hybrids: cell phenotype dependence of virus promoter usage. Virology. 1992 Mar;187(1):189–201. doi: 10.1016/0042-6822(92)90307-b. [DOI] [PubMed] [Google Scholar]
- Khanna R., Burrows S. R., Kurilla M. G., Jacob C. A., Misko I. S., Sculley T. B., Kieff E., Moss D. J. Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development. J Exp Med. 1992 Jul 1;176(1):169–176. doi: 10.1084/jem.176.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klein G. Viral latency and transformation: the strategy of Epstein-Barr virus. Cell. 1989 Jul 14;58(1):5–8. doi: 10.1016/0092-8674(89)90394-2. [DOI] [PubMed] [Google Scholar]
- Laux G., Perricaudet M., Farrell P. J. A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome. EMBO J. 1988 Mar;7(3):769–774. doi: 10.1002/j.1460-2075.1988.tb02874.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lear A. L., Rowe M., Kurilla M. G., Lee S., Henderson S., Kieff E., Rickinson A. B. The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle. J Virol. 1992 Dec;66(12):7461–7468. doi: 10.1128/jvi.66.12.7461-7468.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewin N., Aman P., Masucci M. G., Klein E., Klein G., Oberg B., Strander H., Henle W., Henle G. Characterization of EBV-carrying B-cell populations in healthy seropositive individuals with regard to density, release of transforming virus and spontaneous outgrowth. Int J Cancer. 1987 Apr 15;39(4):472–476. doi: 10.1002/ijc.2910390411. [DOI] [PubMed] [Google Scholar]
- Liu Y. J., Joshua D. E., Williams G. T., Smith C. A., Gordon J., MacLennan I. C. Mechanism of antigen-driven selection in germinal centres. Nature. 1989 Dec 21;342(6252):929–931. doi: 10.1038/342929a0. [DOI] [PubMed] [Google Scholar]
- MacLennan I. C., Gray D. Antigen-driven selection of virgin and memory B cells. Immunol Rev. 1986 Jun;91:61–85. doi: 10.1111/j.1600-065x.1986.tb01484.x. [DOI] [PubMed] [Google Scholar]
- Minarovits J., Hu L. F., Imai S., Harabuchi Y., Kataura A., Minarovits-Kormuta S., Osato T., Klein G. Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas. J Gen Virol. 1994 Jan;75(Pt 1):77–84. doi: 10.1099/0022-1317-75-1-77. [DOI] [PubMed] [Google Scholar]
- Murray R. J., Kurilla M. G., Brooks J. M., Thomas W. A., Rowe M., Kieff E., Rickinson A. B. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J Exp Med. 1992 Jul 1;176(1):157–168. doi: 10.1084/jem.176.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nilsson K., Klein G., Henle W., Henle G. The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV. Int J Cancer. 1971 Nov 15;8(3):443–450. doi: 10.1002/ijc.2910080312. [DOI] [PubMed] [Google Scholar]
- Qu L., Rowe D. T. Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes. J Virol. 1992 Jun;66(6):3715–3724. doi: 10.1128/jvi.66.6.3715-3724.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rocchi G., Felici A., Ragona G., Heinz A. Quantitative evaluation of Epstein-Barr-virus-infected mononuclear peripheral blood leukocytes in infectious mononucleosis. N Engl J Med. 1977 Jan 20;296(3):132–134. doi: 10.1056/NEJM197701202960302. [DOI] [PubMed] [Google Scholar]
- Rowe M., Lear A. L., Croom-Carter D., Davies A. H., Rickinson A. B. Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes. J Virol. 1992 Jan;66(1):122–131. doi: 10.1128/jvi.66.1.122-131.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rowe M., Rowe D. T., Gregory C. D., Young L. S., Farrell P. J., Rupani H., Rickinson A. B. Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. EMBO J. 1987 Sep;6(9):2743–2751. doi: 10.1002/j.1460-2075.1987.tb02568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sample J., Brooks L., Sample C., Young L., Rowe M., Gregory C., Rickinson A., Kieff E. Restricted Epstein-Barr virus protein expression in Burkitt lymphoma is due to a different Epstein-Barr nuclear antigen 1 transcriptional initiation site. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6343–6347. doi: 10.1073/pnas.88.14.6343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sample J., Henson E. B., Sample C. The Epstein-Barr virus nuclear protein 1 promoter active in type I latency is autoregulated. J Virol. 1992 Aug;66(8):4654–4661. doi: 10.1128/jvi.66.8.4654-4661.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schaefer B. C., Woisetschlaeger M., Strominger J. L., Speck S. H. Exclusive expression of Epstein-Barr virus nuclear antigen 1 in Burkitt lymphoma arises from a third promoter, distinct from the promoters used in latently infected lymphocytes. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6550–6554. doi: 10.1073/pnas.88.15.6550. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith P. R., Griffin B. E. Differential expression of Epstein Barr viral transcripts for two proteins (TP1 and LMP) in lymphocyte and epithelial cells. Nucleic Acids Res. 1991 May 11;19(9):2435–2440. doi: 10.1093/nar/19.9.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Su I. J., Lin K. H., Chen C. J., Tien H. F., Hsieh H. C., Lin D. T., Chen J. Y. Epstein-Barr virus-associated peripheral T-cell lymphoma of activated CD8 phenotype. Cancer. 1990 Dec 15;66(12):2557–2562. doi: 10.1002/1097-0142(19901215)66:12<2557::aid-cncr2820661218>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
- Tierney R. J., Steven N., Young L. S., Rickinson A. B. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J Virol. 1994 Nov;68(11):7374–7385. doi: 10.1128/jvi.68.11.7374-7385.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watry D., Hedrick J. A., Siervo S., Rhodes G., Lamberti J. J., Lambris J. D., Tsoukas C. D. Infection of human thymocytes by Epstein-Barr virus. J Exp Med. 1991 Apr 1;173(4):971–980. doi: 10.1084/jem.173.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woisetschlaeger M., Strominger J. L., Speck S. H. Mutually exclusive use of viral promoters in Epstein-Barr virus latently infected lymphocytes. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6498–6502. doi: 10.1073/pnas.86.17.6498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yao Q. Y., Ogan P., Rowe M., Wood M., Rickinson A. B. Epstein-Barr virus-infected B cells persist in the circulation of acyclovir-treated virus carriers. Int J Cancer. 1989 Jan 15;43(1):67–71. doi: 10.1002/ijc.2910430115. [DOI] [PubMed] [Google Scholar]