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Journal of Virology, December 1998, p. 9948-9954, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Residues 231 to 280 of the Epstein-Barr Virus Nuclear Protein 2 Are Not Essential for Primary B-Lymphocyte Growth Transformation

Shizuko Harada, Ramana Yalamanchili, and Elliott Kieff*

Departments of Medicine and Microbiology and Molecular Genetics, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115

Received 9 May 1998/Accepted 8 September 1998

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is a transcriptional transactivator of cellular and viral gene expression and is essential for the transformation of resting human B lymphocytes into long-term lymphoblastoid cell lines (LCLs). Previous molecular genetic analyses identified three domains that are critical for transformation and showed that the rest of EBNA-2 is not critical. We now find that codons 231 to 280 that were part of one of the critical domains (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991) can be deleted with only a small effect on the ability of EBNA-2 to transactivate gene expression. In transient transfection assays, EBNA-2 deleted for codons 231 to 280 accumulated to higher levels and was similar to wild-type EBNA-2 in activation of the BamC promoter and in association with RBPJk, a cellular transcription factor that is important for EBNA-2 interaction with promoter regulatory elements. However, EBNA-2 d231-280 activated the viral latent membrane protein 1 (LMP1) promoter with only 60% of wild-type efficiency. Recombinant EBVs specifically deleted for EBNA-2 codons 231 to 280 were efficient in initiating the transformation of resting primary human B lymphocytes into LCLs. However, these LCLs grew less well than wild-type EBV-transformed LCLs, and 4- to 10-fold more cells were required for outgrowth following limit dilution. EBNA-2 d231-280 accumulated to unusually high levels in the recombinant transformed LCLs, and this was associated with somewhat higher EBNA-1 and lower LMP1 expression, consistent with the near-wild-type activation of the BamC EBNA promoter and the abnormally low activation of the LMP1 promoter in transient transfection assays. Thus, EBNA-2 d231-280 modestly perturbed the regulation of viral gene expression and resulted in less LMP1, while having surprisingly subtle effects on LCL outgrowth. Deletion of EBNA-2 codons 292 to 310, which are closer to the site that specifies interaction with RBPJk, was more disruptive of RBPJk association and of the ability to transform B lymphocytes.

* Corresponding author. Mailing address: Departments of Medicine and Microbiology and Molecular Genetics, Harvard Medical School and Brigham and Women's Hospital, Channing Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4250. Fax: (617) 525-4257. E-mail: ekieff{at}rics.bwh.harvard.edu.

Journal of Virology, December 1998, p. 9948-9954, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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