Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation.
Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.
Cohen
J I
JI
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Wang
F
F
Kieff
E
E
D000595:Amino Acid Sequence; D000944:Antigens, Differentiation, B-Lymphocyte / Q000096:biosynthesis; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics*; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D002472:Cell Transformation, Viral / Q000235:genetics*; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D011951:Receptors, Complement / Q000096:biosynthesis; D017464:Receptors, Complement 3d; D011961:Receptors, Fc / Q000096:biosynthesis; D017455:Receptors, IgE; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins
D000944:Antigens, Differentiation, B-Lymphocyte; D000956:Antigens, Viral; D004279:DNA, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011961:Receptors, Fc; D017455:Receptors, IgE; D014763:Viral Matrix Proteins