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Journal of Virology, October 2007, p. 10924-10932, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.01239-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Replication-Competent Herpes Simplex Virus 1 Isolates Selected from Cells Transfected with a Bacterial Artificial Chromosome DNA Lacking Only the UL49 Gene Vary with Respect to the Defect in the UL41 Gene Encoding Host Shutoff RNase{triangledown}

Maria Teresa Sciortino,1 Brunella Taddeo,2 Maria Giuffrè-Cuculletto,1 Maria Antonietta Medici,1 Antonio Mastino,1 and Bernard Roizman2*

Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone 31, 98166 Messina, Italy,1 The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois 606372

Received 6 June 2007/ Accepted 23 July 2007

To generate a null UL49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the UL49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-{Delta}UL49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the UL41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-UL49R DNA (R-UL49) accumulated a full-length vhs protein, indicating that in the parental BAC-{Delta}UL49 DNA, the UL41 gene was intact. We conclude that expression of the vhs protein in the absence of UL49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of UL49, to be neutralized.

* Corresponding author. Mailing address: University of Chicago, Viral Oncology Laboratory, 910 East 58th St., Chicago, IL 60637. Phone: (773) 702-1898. Fax: (773) 702-1631. E-mail: bernard.roizman{at}bsd.uchicago.edu

{triangledown} Published ahead of print on 1 August 2007.

Journal of Virology, October 2007, p. 10924-10932, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.01239-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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