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Journal of Virology, February 2000, p. 1663-1673, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Isolation of Herpes Simplex Virus Procapsids from Cells Infected with a Protease-Deficient Mutant Virus

William W. Newcomb,1 Benes L. Trus,2,3 Naiqian Cheng,2 Alasdair C. Steven,2 Amy K. Sheaffer,4 Daniel J. Tenney,4 Sandra K. Weller,5 and Jay C. Brown1,*

Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 229081; Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases,2 and Computational Bioscience and Engineering Laboratory, Center for Information Technology,3 National Institutes of Health, Bethesda, Maryland 20892; Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 064924; and Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 060305

Received 1 September 1999/Accepted 10 November 1999

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids---normally, short-lived intermediates---would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0°C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-Å resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.

* 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}VIRGINIA.EDU.

Journal of Virology, February 2000, p. 1663-1673, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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