The antibiotic tunicamycin, which blocks the synthesis of glycoproteins, inhibited the production of infectious herpes simplex virus. In the presence of this drug, ['4C]glucosamine and [3H]mannose incorporation was reduced in infected cells, whereas total protein synthesis was not affected. Gel electrophoresis of [2-3H]mannose-labeled polypeptides failed to detect glycoprotein D or any of the other herpes simplex virus glycoproteins. By use of specific antisera we demonstrated that in the presence of tunicamycin the normal precursors to viral glycoproteins failed to appear. Instead, lower-molecular-weight polypeptides were found which were antigenically and structurally related to the glycosylated proteins. Evidence is presented to show that blocking the addition of carbohydrate to glycoprotein precursors with tunicamycin results in the disappearance of molecules, possibly due to degradation of the unglycosylated polypeptides. We infer that the added carbohydrate either stabilizes the envelope proteins or provides the proper structure for correct processing of the molecules needed for infectivity. The contribution of the carbohydrate portion of viral glycoproteins to infectivity, antigenicity,

Primary cultures of rat and mouse sensory neurons were used to study the entry of herpes simplex virus type 1 (HSV-1). Soluble, truncated nectin-1 but not HveA prevented viral entry. Antibodies against nectin-1 also blocked infection of rat neurons. These results indicate that nectin-1 is the primary receptor for HSV-1 infection of sensory neurons.Herpes simplex virus type 1 (HSV-1) has a broad host range, and recent studies have identified a number of cell surface proteins that serve as receptors for viral entry (reviewed in reference 18). It appears that the abundance of the different receptors varies with the cell type, and this variation might influence the course of HSV-1 infection (4). The envelope of HSV-1 contains several glycoproteins, and studies with mutant virus have shown that the glycoproteins gB, gD, and gH are required for infection of rat neurons (1). An essential event in virus cell interaction is the binding of viral glycoprotein D (gD) to a cellular receptor. Glycoprotein D interacts with at least three structurally unrelated receptors: HveA (12, 13, 21), nectin-1 (4, 7), and 3-O-sulfated heparan sulfate (16). HveA, also known as HVEM, is a member of the tumor necrosis factor receptor superfamily. HveA mRNA is expressed in lymphoid cells and fibroblasts but only weakly in human brain tissue (8,12). Nectin-1, also called HveC, is a member of the immunoglobulin superfamily. Nectin-1 is found at cellular junctions and is involved in cell-cell adhesion (14, 19) and in synapse formation (11). High levels of nectin-1 mRNA are expressed in the human central nervous system (2), in neuronal cell lines (4), and in mouse sensory, sympathetic, and parasympathetic neurons (5). Nectin-1 protein is found in abundance in rat sensory neurons but not in rat motor neurons (9). Wilcox and Johnson developed a model of HSV-1 latency in primary sensory neurons (22,23). This model reproduces many of the characteristic features of a natural human HSV-1 latent infection, including restricted viral gene expression (3) and reactivation, to produce infectious virus (17,23). Using this model and functional assays to measure virus entry, evidence was obtained that HSV-1 entry into rodent sensory neurons is mediated by nectin-1.Effects of soluble HveA or nectin-1 receptors on HSV-1 entry. Neuronal cultures were prepared from dorsal root ganglia of embryonic day 15 rats or mice as previously described (22, 23). Dulbecco's Eagle's medium-F12 (supplemented with 10% newborn bovine serum, 100 ng of 2.5-S mouse nerve growth factor/ml, and 20 M 5-fluoro-2Ј-deoxyuridine to inhibit growth of nonneuronal cells) was used to establish neuronal cultures (neuronal maintenance medium). Rats and mice were treated according to institutional guidelines for animal use. Primary rat fibroblasts and HeLa cells were cultured in Dulbecco's Eagle's medium with 5% fetal bovine serum. For infection, a recombinant HSV-1 (17 ϩ strain) expressing green fluorescent protein (GFP) fused to the C terminus of the immediate-early gene product ICP4 was used....

The amino acid analogue L-serine hydroxamate, which is bacteriostatic for Escherichia coli, has been shown to inhibit protein synthesis. The antimetabolite is a competitive inhibitor of seryl-transfer ribonucleic acid (tRNA) synthetase with a K; value of 30 uM. Mutants resistant to L-serine hydroxamate have been selected, and three were shown to have seryl-tRNA synthetases with increased K, values. One mutant contains a 3-phosphoglycerate dehydrogenase which is insensitive to inhibition by L-serine.

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In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that (i) specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and (il) ICP4 stimulates initiation of transcription from thegD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.Herpes simplex virus (HSV) proteins synthesized in infected cells change in both number and character during productive infection (1,2 We have used an in vitro transcription system (27) to investigate how a partially purified preparation of the viral protein ICP4 interacts with DNA from the early HSV gene for glycoprotein D (gD). In this paper we present evidence that ICP4 binds specifically to DNA sequences adjacent to the gD gene promoter and stimulates accurate transcription from this early gene. The mechanism of stimulation by ICP4 involves an increase in initiation of RNA synthesis. This report identifies a specific step in the transcription process that is regulated by an HSV protein.MATERIALS AND METHODS Template DNA. The HSV DNA used in this study was prepared from the plasmid pJB3. This plasmid contains the Sma I fragment subcloned from the BamHI fragment J of HSV type 1 (HSV-1) (KOS). The construction pJB3 and a simplified restriction map of the Sma I fragment are shown in Fig. 1. More details on the plasmid and its use in mapping the gD mRNA are presented in an earlier publication (36). To obtain the Ava I fragment 1 for use in the in vitro transcription reactions, plasmid DNA was purified by two cycles of cesium chloride centrifugation, cut with the restriction enzyme Ava I (Bethesda Research Laboratories), and extensively extracted with phenol/chloroform, 1:1 (vol/vol). The DNA fragments were precipitated with ethanol, redissolved in buffer, and separated by electrophoresis on 1% agarose gels. The 1.55-kilobase-pair (kbp) Ava I fragment 1 was isolated by electrophoresis into a block of low-temperature-gelling agarose, application of heat to 680C, extraction with phenol, and precipitation with alcohol. The DNA fragment was dissolved in 10 mM Tris chloride, pH 7.5/1 mM EDTA and was used directly as template for in vitro transcription.The Sst I (Sac I) subclone of pJB3 was constructed by inserting the Sst I fragment that contains the gD gene into the unique ...