A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells

doi:10.1128/JVI.74.24.11608-11618.2000

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Journal of Virology, December 2000, p. 11608-11618, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells

Prashant J. Desai^*

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 18 July 2000/Accepted 18 September 2000

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designatedVP1/2, which is also a component of the virion tegument. A nullmutation was generated in the UL36 gene to elucidate its rolein the virus life cycle. Since the UL36 gene specifies an essentialfunction, complementing cell lines transformed for sequences encodingthe UL36 ORF were made. A mutant virus, designated K $Delta$ UL36, thatencodes a null mutation in the UL36 gene was isolated and propagatedin these cell lines. When noncomplementing cells infected withK $Delta$ UL36 were analyzed, both terminal genomic DNA fragments andDNA-containing capsids (C capsids) were detected; therefore, UL36is not required for cleavage or packaging of DNA. Sedimentationanalysis of lysates from mutant-infected cells revealed the presenceof particles that have the physical characteristics of C capsids.In agreement with this, polypeptide profiles of the mutant particlesrevealed an absence of the major envelope and tegument components.Ultrastructural analysis revealed the presence of numerous unenvelopedDNA containing capsids in the cytoplasm of K $Delta$ UL36-infected cells.The UL36 mutant particles were tagged with the VP26-green fluorescentprotein marker, and their movement was monitored in living cells.In K $Delta$ UL36-infected cells, extensive particulate fluorescence correspondingto the capsid particles was observed throughout the cytosol. Accumulationof fluorescence at the plasma membrane which indicated maturationand egress of virions was observed in wild-type-infected cellsbut was absent in K $Delta$ UL36-infected cells. In the absence of UL36function, DNA-filled capsids are produced; these capsids enterthe cytosol after traversing the nuclear envelope and do not matureinto enveloped virus. The maturation and egress of the UL36 mutantparticles are abrogated, possibly due to a late function of thiscomplex polypeptide, i.e., to target capsids to the correct maturationpathway.

^* Mailing address: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. WolfeSt., Baltimore, MD 21205. Phone: (410) 614-1581. Fax: (410) 955-3023.E-mail: pdesai{at}jhmi.edu.

Journal of Virology, December 2000, p. 11608-11618, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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