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Journal of Virology, May 1999, p. 4239-4250, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Assembly of the Herpes Simplex Virus Procapsid from Purified Components and Identification of Small Complexes Containing the Major Capsid and Scaffolding Proteins

William W. Newcomb,1 Fred L. Homa,2 Darrell R. Thomsen,2 Benes L. Trus,3,4 Naiqian Cheng,3 Alasdair Steven,3 Frank Booy,5 and Jay C. Brown1,*

Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 229081; Infectious Disease Research, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 490012; and Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases,3 and Computational Bioscience and Engineering Laboratory, Center for Information Technology,4 National Institutes of Health, and Biomedical Engineering and Instrumentation Program, National Center for Research Resources,5 Bethesda, Maryland 20892

Received 23 November 1998/Accepted 9 February 1999

An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ~30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

* Corresponding author. Mailing address: Department of Microbiology, Box 441, University of Virginia Health Sciences Center, Charlottesville, VA 22908. Phone: (804) 924-1814. Fax: (804) 982-1071. E-mail: JCB2G{at}AVERY.MED.VIRGINIA.EDU.

Journal of Virology, May 1999, p. 4239-4250, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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