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Journal of Virology, July 2002, p. 7349-7355, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.7349-7355.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The EBNA2 Polyproline Region Is Dispensable for Epstein-Barr Virus-Mediated Immortalization Maintenance

Alexey V. Gordadze, David Poston, and Paul D. Ling*

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030

Received 15 January 2002/ Accepted 18 April 2002

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 {Delta}PPR-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 {Delta}PPR-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 {Delta}PPR-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 {Delta}PPR-EBNA2 EBVs to immortalize B cells.

* Corresponding author. Mailing address: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Mail Stop no. BCM-385, 1 Baylor Plaza, Houston, Texas 77030. Phone: (713) 798-8474. Fax: (713) 798-3586. E-mail: pling{at}bcm.tmc.edu.

Journal of Virology, July 2002, p. 7349-7355, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.7349-7355.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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