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Journal of Virology, March 2002, p. 2480-2490, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2480-2490.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Separation of the DNA Replication, Segregation, and Transcriptional Activation Functions of Epstein-Barr Nuclear Antigen 1

*** Hong Wu,1 Priya Kapoor,1 and Lori Frappier1*

Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A81

Received 16 August 2001/ Accepted 12 November 2001

In latent Epstein-Barr virus infection, the viral EBNA1 protein binds to specific sites in the viral origin of DNA replication, oriP, to activate the initiation of DNA replication, enhance the expression of other viral latency proteins, and partition the viral episomes during cell division. The DNA binding domain of EBNA1 is required for all three function, and a Gly-Arg-rich sequence between amino acids 325 and 376 is required for both the transcriptional activation and partitioning functions. We have used mutational analysis to identify additional EBNA1 sequences that contribute to EBNA1 functions. We show that EBNA1 amino acids 8 to 67 contribute to, but are not absolutely required for, EBNA1 replication, partitioning, and transcriptional activation functions. A Gly-Arg-rich sequence (amino acids 33 to 53) that is similar to that of amino acids 325 to 376 and lies within the 8-to-67 region was not responsible for the functional contributions of residues 8 to 67, since deletion of amino acids 34 to 52 alone did not affect EBNA1 functions. We also found that deletion of amino acids 61 to 83 eliminated the transcriptional activity of EBNA1 without affecting partitioning. This mutant also exhibited an increased replication efficiency that resulted in the maintenance of oriP plasmids at a copy number approximately fourfold higher than for wild-type EBNA1. The results indicate that the three EBNA1 functions have overlapping but different sequence requirements. Transcriptional activation requires residues 61 to 83 and 325 to 376 and is stimulated by residues 8 to 67; partitioning requires residues 325 to 376 and is stimulated by residues 8 to 67; and replication involves redundant contributions of both the 325-to-376 and 8-to-67 regions.

* Corresponding author. Mailing address: Department of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8. Phone: (416) 946-3501. Fax: (416) 978-6885. E-mail: lori.frappier{at}utoronto.ca.

Journal of Virology, March 2002, p. 2480-2490, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2480-2490.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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