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J. Biol. Chem., Vol. 279, Issue 39, 40358-40361, September 24, 2004

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The Epstein-Barr Virus Polymerase Accessory Factor BMRF1 Adopts a Ring-shaped Structure as Visualized by Electron Microscopy^*

Alexander M. Makhov, Deepa Subramanian, Elizabeth Holley-Guthrie, Shannon C. Kenney, and Jack D. Griffith¶

From the Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center and Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7295

Epstein-Barr virus (EBV) encodes a set of core replication factors used during lytic infection in human cells that parallels the factors used in many other systems. These include a DNA polymerase and its accessory factor, a helicase/primase, and a single strand binding protein. The EBV polymerase accessory factor has been identified as the product of the BMRF1 gene and has been shown by functional assays to increase the activity and processivity of the polymerase. Unlike other members of this class of factors, BMRF1 is also a transcription factor regulating certain EBV genes. Although several polymerase accessory factors, including eukaryotic proliferating cell nuclear antigen, Escherichia coli protein, and T4 gene 45 protein have been shown to form oligomeric rings termed sliding clamps, nothing is known about the oligomeric state of BMRF1 or whether it forms a ring. In this work, BMRF1 was purified directly from human cells infected with an adenovirus vector expressing the BMRF1 gene product. The protein was purified to near homogeneity, and examination by negative staining electron microscopy revealed large, flat, ring-shaped molecules with a diameter of 15.5 ± 0.8 nm and a distinct 5.3-nm diameter hole in the center. The size of these rings is consistent with an oligomer of 6 monomers, nearly twice as large as the trimeric proliferating cell nuclear antigen ring. Unlike the herpes simplex virus UL42 homologue, BMRF1 was found to self-associate in solution. These findings extend the theme of polymerase accessory factors adopting ring-shaped structures and provide an example in which the ring is significantly larger than any previously described sliding clamp.

Received for publication, July 30, 2004

^* This work was supported by National Institutes of Health Grants CA19014 (to J. D. G. and S. C. K.) and GM31819 (to J. D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 919-966-2151; Fax: 919-966-3015; E-mail: jdg{at}med.unc.edu.

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