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Journal of Virology, January 2005, p. 245-256, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.245-256.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Epstein-Barr Virus Replication Protein BBLF2/3 Provides an Origin-Tethering Function through Interaction with the Zinc Finger DNA Binding Protein ZBRK1 and the KAP-1 Corepressor

Gangling Liao,¹ Jian Huang,² Elizabeth D. Fixman,^2, and S. Diane Hayward^1,2*

Sidney Kimmel Comprehensive Cancer Center,¹ Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland²

Received 8 June 2004/ Accepted 12 August 2004

Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (ZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.

Present address: Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Canada.

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