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

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-AU_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-AU_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.