Rebecca I. Montgomery

Herpes Simplex Virus-1 Entry into Cells Mediated by a Novel Member of the TNF/NGF Receptor Family

We identified and cloned a cellular mediator of herpes simplex virus (HSV) entry. Hamster and swine cells resistant to viral entry became susceptible upon expression of a human cDNA encoding this protein, designated HVEM (for herpesvirus entry mediator). HVEM was shown to mediate the entry of several wild-type HSV strains of both serotypes. Anti-HVEM antibodies and a soluble hybrid protein containing the HVEM ectodomain inhibited HVEM-dependent infection but not virus binding to cells. Mutations in the HSV envelope glycoprotein gD significantly reduced HVEM-mediated entry. The contribution of HVEM to HSV entry into human cells was demonstrable in activated T cells. HVEM, the first identified mediator of HSV entry, is a new member of the TNF/NGF receptor family.

LIGHT, a New Member of the TNF Superfamily, and Lymphotoxin α Are Ligands for Herpesvirus Entry Mediator

Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin alpha (LTalpha) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTalphabeta heterotrimer but does not form complexes with either LTalpha or LTbeta. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.

Contributions of gD receptors and glycosaminoglycan sulfation to cell fusion mediated by herpes simplex virus 1

Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.

Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly

With the exception of hyaluronic acid, all mammalian saccharides assemble while attached to a lipid or protein primer. Several cases are now known in which oligosaccharide synthesis will occur on synthetic glycoside primers added to cells. A protocol is described in this unit in which b‐D‐xylosides initiate glycosaminoglycan (GAG) synthesis by substituting for endogenous xylosylated core proteins. At high concentration xylosides will also prime oligosaccharides that resemble glycolipids. N‐acetyl‐a‐D‐galactosaminides initiate the synthesis of O‐linked oligosaccharides found on mucins and other glycoproteins in an analogous manner. Even disaccharides, such as peracetylated N‐acetyllactosaminide, can act as primers. Because these primers compete with endogenous substrates, they also act as inhibitors of proteoglycan (PG) and glycoprotein synthesis. Thus, primers have utility for studying the biological activity of glycoconjugates in cells, tissues, and animals. This unit describes procedures for using glycoside primers in cell culture.