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.

No abstract

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 ...

and Beltchev, 1988) and recombination (Bianchi et al., USA 1989;Paull et al., 1993). Central to all these processes 1 Present address: Institute of Microbiology, Centre Hospitalier may be the ability of HMG to increase DNA flexibility Universitaire Vaudois, 1011 Lausanne, Switzerland and promote the formation of nucleoprotein complexes. 4 Corresponding author HMG1 bends DNA without sequence specificity. It has E.Costello and P.Saudan contributed equally to this work. been shown to enable the ligation to circular forms of short DNA fragments which are otherwise too inflexible High mobility group protein 1 (HMG1) is an abundant for self-ligation (Pil et al., 1993;Paull et al., 1993). non-histone chromosomal protein which plays a role Onate et al. (1994) observed enhanced binding of the in several nuclear events involving DNA. Here we progesterone receptor to its DNA recognition element in demonstrate that HMG1 physically interacts with the the presence of HMG1 and, having found that HMG1 is human adeno-associated virus (AAV) Rep protein.an effective DNA-flexing protein, proposed a structural HMG1 promotes the formation of Rep-DNA complexes alteration of the progesterone receptor DNA target site as and stimulates the activity of Rep in site-and stranda possible mechanism for the enhanced binding. However, specific cleavage of DNA and the hydrolysis of ATP, increasing DNA flexibility is not the sole means by which functions required for viral gene regulation, replication HMG may influence cellular processes. By physically and site-specific integration of viral DNA into human interacting with TATA-binding protein, HMG1 indirectly chromosome 19. We show that HMG1 enhances Repinhibited class II gene transcription (Ge and Roeder, 1994). mediated repression of the AAV p5 promoter in transStimulation of transcription by HMG1 has also been fected cells, suggesting that HMG1 and Rep also reported (Ogawa et al., 1995). Recently, Zappavigna et al. interact in vivo. HMG1, Rep and DNA can be immuno- (1996) showed that HMG1 interacts with the HOXD9 precipitated as a ternary complex. Kinetic studies protein and enhances its sequence-specific DNA-binding indicate that complexes of Rep with DNA have similar activity in vitro. HMG1 stimulated transcriptional activstabilities in the presence and absence of HMG1.These ation by HOXD9 in vivo. The closely related HMG2 was results suggest that the effect of HMG1 on Rep binding isolated in a screening for proteins which interact with is exerted at the step of complex formation and thereby Oct2 (Zwilling et al., 1995). HMG2 increased specific may reflect an activity of HMG1 in promoting the DNA binding by Oct2 and stimulated Oct2-dependent assembly of complex cellular nucleoprotein structures.transcription.

To determine the type of cell(s) that contain latent varicella-zoster virus (VZV) DNA, we prepared pure populations of neurons and satellite cells from trigeminal ganglia of 18 humans who had previously had a VZV infection. VZV DNA was present in 34 of 2,226 neurons (1.5%) and in none of 20,700 satellite cells. There was an average of 4.7 (range of 2 to 9) copies of VZV DNA per latently infected neuron. Latent VZV DNA was primarily present in large neurons, whereas the size distribution of herpes simplex virus DNA was markedly different.Varicella-zoster virus (VZV) is present in a latent form in sensory ganglia of humans who have had a primary infection (varicella) with VZV (11). This is demonstrated by the reactivation of VZV in these individuals as herpes zoster and by the presence of VZV DNA and proteins in ganglia recovered at autopsy (2,3,5,9,10,12,13,15,16,19,20,(22)(23)(24)26). Different laboratories have ascribed the site of latency to either neurons (12-14, 18, 22) or perineuronal satellite cells (5, 26); some laboratories suggest that neurons are the primary site of latency together with a smaller proportion of satellite cells containing the VZV genome (16,20). The primary aim of the experiments described here was to determine the type of cell in trigeminal ganglia that harbors latent VZV and also to obtain data on the proportion of these cells that contain latent VZV. One reason why the site of VZV latency has remained an open question is that neurons and satellite-supporting cells are tightly associated in ganglia and the in situ methods used to locate VZV DNA did not clearly resolve the source of the hybridization signal. This resolution was easier for latent HSV because a strong hybridization signal to HSV facilitated its localization to the nucleus of the neuron. In contrast, the hybridization signal from VZV DNA is considerably weaker and dispersed over both nucleus and cytoplasm. While the hybridization methodology has improved, these considerations remain (21, 30). To reduce the ambiguities inherent in the in situ methods, we developed a procedure to separate cell types prior to analysis. The separation procedure has been verified with a study of HSV (1). This report presents data on the cellular localization of latent VZV, the frequency of latency, and the number of viral genomes present in latent cells.

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with 32P-inorganic phosphate and 32P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.