title: Residues 231 to 280 of the Epstein-Barr virus nuclear protein 2 are not essential for primary B-lymphocyte growth transformation.
abstract: 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.
authors.affiliation: Departments of Medicine and Microbiology and Molecular Genetics, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: S
authors.initials: S
authors.lastname: Harada
authors.forename: R
authors.initials: R
authors.lastname: Yalamanchili
authors.forename: E
authors.initials: E
authors.lastname: Kieff
mesh_terms: D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000235:genetics* / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics* / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014763:Viral Matrix Proteins / Q000235:genetics / Q000502:physiology
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009687:Nuclear Proteins; C497570:RBPJ protein, human; D014763:Viral Matrix Proteins