The EBNA2 polyproline region is dispensable for Epstein-Barr virus-mediated immortalization maintenance.
Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that DeltaPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing DeltaPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in DeltaPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant DeltaPPR-EBNA2 EBVs to immortalize B cells.
Gordadze
Alexey V
AV
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Poston
David
D
Ling
Paul D
PD
D001402:B-Lymphocytes / Q000821:virology*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003470:Culture Media; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry* / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D003470:Culture Media; C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D004967:Estrogens; D010455:Peptides; D014764:Viral Proteins; C011083:polyproline