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J Virol, February 1998, p. 1060-1070, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

Alistair R. McNab,1 Prashant Desai,2 Stan Person,2 Lori L. Roof,1 Darrell R. Thomsen,1 William W. Newcomb,3 Jay C. Brown,3 and Fred L. Homa1,*

Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 490071; Virology Laboratories, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 212052; and Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 229083

Received 16 September 1997/Accepted 29 October 1997

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.

* Corresponding author. Mailing address: Pharmacia & Upjohn, Inc., 7242-267-507, 301 Henrietta, Kalamazoo, MI 49007. Phone: (616) 833-9724. Fax: (616) 833-2599. E-mail: fred.l.homa{at}am.pnu.com.




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