Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.

Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI grew to lower titers in melanoma cells than did parental VZV. While VZV deleted for gI replicated in other human cells, the mutant virus replicated to very low titers in primary guinea pig and monkey cells and did not replicate in Vero cells. When compared with the parental virus, rescued viruses, in which the gI deletion was restored with a wild-type allele, showed a similarly sized gE and comparable growth patterns in melanoma and Vero cells. VZV deleted for gI entered Vero cells; however, viral DNA synthesis was impaired in these cells. The VZV gI mutant was slightly impaired for adsorption to human cells. Thus, VZV gI is required for replication of the virus in Vero cells, for efficient replication of the virus in nonhuman cells, and for normal processing of gE.     

Chemical structure of nuclear proteins which are phosphorylated during meiotic maturation of starfish oocytes

Oocytes of the starfish, Asterina pectinifera, are arrested at the G2 phase of meiosis I and possess a prominent germinal vesicle in which maternal stores of nuclear proteins which are destined for use primarily by the early embryo are stored. Germinal vesicle breakdown and subsequent oocyte maturation is triggered by activation of the p34(cdc2)/cyclin B complex, which is present as the preform in the cytoplasm. The aim of the present study was to identify and biochemically characterize in vivo substrates of the kinase. Two nucleic acid binding nuclear proteins designated NAAP1 and NAAP2 were found, both of which contain 345 amino acid residues with pI 3. 6 and which serve as substrates. The only difference between the two proteins was in the primary amino acid sequence at position 51, which is Asn in NAAP1 but Thr in NAAP2. NAAPs are phosphorylated in vivo during oocyte maturation but not at the meiotic G(2) stage. NAAPs are phosphorylated in vitro by the cdc2 kinase on the same site as in vivo. Although there are other evolutionarily conserved consensus sequences for phosphorylation by mitotically active cdc2 kinase in NAAPs and NAAP-derived fragments containing the sequences were efficiently phosphorylated in vitro, these sites in the intact NAAPs were not phosphorylated either in vivo or in vitro. These results suggest that the tertiary structure of NAAPs affects the target specificity of the cdc2 kinase.     

Formation and rearrangement of disulfide bonds during maturation of the Sindbis virus E1 glycoprotein.

The rigidly ordered icosahedral lattice of the Sindbis virus envelope is composed of a host-derived membrane bilayer in which the viral glycoproteins E1 and E2 reside. E1-E1 interactions stabilized by intramolecular disulfide bridges play a significant role in maintaining the envelope's structural integrity (R. P. Anthony and D. T. Brown, J. Virol. 65:1187-1194, 1991; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have examined the acquisition of disulfide bridges within E1 during its maturation. Prior to exit from the endoplasmic reticulum, E1 folds via at least three intermediates, differing in the number and/or arrangement of their disulfides, into a single, compact form. This E1 species remains stable with respect to its disulfides until late in the secretory pathway, when E1 attains a metastable conformation. At this point, when appropriately triggered, intramolecular thiol-disulfide exchange reactions within E1 can occur, resulting in the generation of alternative E1 species. This metastable nature of mature E1 may have important implications for the mechanism of virus disassembly during the initial stages of the infection process (B. Abell and D. T. Brown, J. Virol. 67:5496-5501, 1993).     

Isolation and characterization of phosphorylated oligosaccharides from alpha-N-acetylglucosaminidase that are recognized by cell-surface receptors.

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6-phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026-2030; Ullrich et al. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1591-1598]. Loss of alpha-N-acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of alpha-N-acetylglucosaminidase is located on N-glycosidically linked oligosaccharides of the high mannose type. Acidic oligosaccharides with an average molecular weight of 2200 were liberated from alpha-N-acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, alpha-mannosidase and beta-N-acetylglucosaminidase. At the non-reducing terminal these oligosaccharides bear phosphorylated mannose and/or N-acetylglucosamine residues.     

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type 1.

Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide.     

Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus.

Intact Sindbis virus and Triton-solubilized viral glycoprotein were treated with alpha-mannosidase and with a preparation of mixed glycosidases from Diplococcus pneumoniae to probe the accesibility of carbohydrate units on the viral surface. The products of glycosidase attack on Triton-solubilized virus showed that mose carbohydrate units of the glycoproteins are good substrates for these enzymes. The relative resistance of most of the viral oligosaccharides in intact virus particles showed that much of the carbohydrate is not accessible to glycosidases, probably because it is not exposed at the viral surface. The only completely accessible carbohydrate units on Sindbis glycoproteins were the type A oligosaccharides of E2. This differential accessibility of Sindbis oligosaccharides is discussed in relation to the organization of the viral surface.     

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.     

Varicella-zoster virus Fc receptor gE glycoprotein: serine/threonine and tyrosine phosphorylation of monomeric and dimeric forms.

Varicella-zoster virus (VZV) glycoprotein gE is the predominant viral cell surface molecule; it behaves as an Fc receptor for immunoglobulin G, but its central function may be more closely related to viral egress and cell-to-cell spread. To further analyze the receptor properties of VZV gE, the gE gene (also called open reading frame 68) was expressed by a baculovirus vector in insect cells. The recombinant baculovirus gE product had a molecular mass of 64 kDa, smaller than the previously documented 98 kDa of mature gE expressed in mammalian cells. The major reason for the lowered molecular mass was diminished glycosylation. In addition to the 64-kDa form, a larger (130-kDa) form was observed in insect cells and represented dimerized 64-kDa molecules. Both the monomeric and dimeric gE forms were highly phosphorylated in insect cells. Protein kinase assays conducted in vitro with [gamma-32P]ATP and [gamma-32P]GTP indicated that endogenous casein kinase II was phosphorylating monomeric gE, while the dimeric gE form was phosphorylated by another kinase which did not utilize [gamma-32P]GTP. When immobilized recombinant gE molecules were probed with a monoclonal antibody which specifically recognizes a phosphotyrosine linkage, the gE dimer was found to be tyrosine phosphorylated whereas the monomer was not similarly modified. When recombinant gE produced in HeLa cells was probed with the same antiphosphotyrosine antibody, a dimeric gE form at 130 kDa was detected on the cell surface. These results suggested that VZV gE closely resembled other cell surface receptors, being modified on its various forms by both serine/threonine and tyrosine protein kinases. In this case, tyrosine phosphorylation occurred on a previously unrecognized and underglycosylated VZV gE dimeric product.     

Varicella-zoster virus vaccine DNA differs from the parental virus DNA.

The DNAs of a varicella-zoster virus vaccine and its parental virus were compared by CsCl buoyant density centrifugation and restriction enzyme cleavage analysis. The varicella-zoster virus vaccine DNA showed a heterogeneous buoyant profile and altered restriction enzyme cleavage patterns. These changed properties are probably the result of the accumulation of virus containing defective varicella-zoster virus DNA during extensive cell culture passage of the vaccine virus.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells

Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase, azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthetic maturation of an ascites tumor cell surface sialomucin. Evidence for O-glycosylation of cell surface glycoprotein by the addition of new oligosaccharides during recycling

Previous biosynthetic studies of the ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 (ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the cell surface in 2 h. Since transit to the cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the cell surface. Initiation of new oligosaccharides on cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.     

Signal peptide requirements for lymphocytic choriomeningitis virus glycoprotein C maturation and virus infectivity

Insertion of the lymphocytic choriomeningitis virus (LCMV) precursor glycoprotein C (GP-C) into the membrane of the endoplasmic reticulum is mediated by an unusual signal peptide (SP(GP-C)). It is comprised of 58 amino acid residues and contains an extended hydrophilic N-terminal region, two hydrophobic regions, and a short C-terminal region. After cleavage by signal peptidase, SP(GP-C) accumulates in cells and virus particles. In the present study, we identified the LCMV SP(GP-C) as being an essential component of the GP complex and show that the different regions of SP(GP-C) are required for distinct steps in GP maturation and virus infectivity. More specifically, we show that one hydrophobic region of SP(GP-C) is sufficient for the membrane insertion of GP-C, while both hydrophobic regions are required for the processing and cell surface expression of the GPs. The N-terminal region of SP(GP-C), on the other hand, is essential for pseudoviral infection of target cells. Furthermore, we show that unmyristoylated SP(GP-C) exposes its N-terminal region to the exoplasmic side. This SP(GP-C) can promote GP-C maturation but is defective in pseudoviral infection. Myristoylation and topology of SP(GP-C) in the membrane may thus hold the key to an understanding of the role of SP(GP-C) in GP-C complex maturation and LCMV infectivity.     

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).     

Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides.

Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers; (ii) the rate of virus entry into uninfected cells; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.

Complex asparagine (N)-linked oligosaccharides appear late in phylogeny and are highly regulated in vertebrates. Variations in these structures are found on the majority of cell-surface and secreted proteins. Complex N-linked oligosaccharide biosynthesis is initiated in the Golgi apparatus by the action of Mgat-1-encoded UDP-N-acetylglucosamine:alpha-3-D- mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI). To determine if these structures govern ontogenic processes in mammals, mouse embryos were generated that lacked a functional Mgat-1 gene. Inactivation of both Mgat-1 alleles produced deficiencies in GlcNAc-TI activity and complex N-linked oligosaccharides. Embryonic lethality occurred by day 10.5, thus establishing that complex N-linked oligosaccharides are required during post-implantation development. Remarkably, embryonic development proceeded into day 9 with the differentiation of multiple cell types. Complex N-linked oligosaccharides are important for morphogenic processes as neural tube formation, vascularization and the determination of left-right body plan asymmetry were impaired in the absence of a functional Mgat-1 gene.