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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 U_L49 Gene Vary with Respect to the Defect in the U_L41 Gene Encoding Host Shutoff RNase

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 U_L49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U_L49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-U_L49 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 U_L41 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-U_L49R DNA (R-U_L49) accumulated a full-length vhs protein, indicating that in the parental BAC-U_L49 DNA, the U_L41 gene was intact. We conclude that expression of the vhs protein in the absence of U_L49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U_L49, to be neutralized.

Published ahead of print on 1 August 2007.

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