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Journal of Virology, September 2003, p. 9862-9871, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9862-9871.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

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

Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908,1 Infectious Disease Research, Pharmacia Corporation, Kalamazoo, Michigan 490012

Received 17 April 2003/ Accepted 18 June 2003

The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 UL6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, UL26.5. Soluble UL26.5-portal complexes were found to assemble when purified portals were mixed in vitro with UL26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter "scaffold particles" formed from purified UL26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and UL26.5 combine in various proportions, with the highest observed UL6 content corresponding to two or three portals per scaffold particle. Association between the portal and UL26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including UL6, at a level corresponding to ~1 portal complex per capsid. The results support the view that UL6 becomes incorporated into nascent HSV-1 capsids by way of a complex with UL26.5 and suggest further that UL6 may be introduced into the growing capsid as an intact portal.

* 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, September 2003, p. 9862-9871, Vol. 77, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.18.9862-9871.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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