Replication-Competent Herpes Simplex Virus 1 Isolates Selected from Cells Transfected with a Bacterial Artificial Chromosome DNA Lacking Only the U L 49 Gene Vary with Respect to the Defect in the U L 41 Gene Encoding Host Shutoff RNase

To generate a null UL 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the UL 49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-ΔUL 49 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 UL 41 gene. One mutant virus expressed novhs protein, whereas two others, derived from independent transfections, produced truncatedvhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-UL 49R DNA (R-UL 49) accumulated a full-lengthvhs protein, indicating that in the parental BAC-ΔUL 49 DNA, the UL 41 gene was intact. We conclude that expression of thevhs protein in the absence of UL 49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cellsvhs requires both VP16 and VP22, the product of UL 49, to be neutralized.