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Journal of Virology, July 2006, p. 6286-6294, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02648-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Herpes Simplex Virus Capsid Structure: DNA Packaging Protein UL25 Is Located on the External Surface of the Capsid near the Vertices

William W. Newcomb,1 Fred L. Homa,2 and Jay C. Brown1*

Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908,1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152612

Received 19 December 2005/ Accepted 6 April 2006

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.

* Corresponding author. Mailing address: Department of Microbiology, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Phone: (434) 924-1814. Fax: (434) 982-1071. E-mail: jcb2g{at}virginia.edu.

Journal of Virology, July 2006, p. 6286-6294, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02648-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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