The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor kappa B (NF-kappa B) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-kappa B activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-kappa B activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-kappa B activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-kappa B activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-kappa B activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-kappa B activation by HSV-1 gD.

ABSTRACT To generate a null U L 49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U L 49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-ΔU L 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 U L 41 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 L 49R DNA (R-U L 49) accumulated a full-length vhs protein, indicating that in the parental BAC-ΔU L 49 DNA, the U L 41 gene was intact. We conclude that expression of the vhs protein in the absence of U L 49 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 L 49, to be neutralized.