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1.
A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo- or hetero-oligomers. Purified KOS was incubated at 4 degrees C with bis (sulfosuccinimidyl) suberate (BS3), an 11.4 A (1A = 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo- and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes. 相似文献
2.
Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface. 总被引:12,自引:9,他引:3
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Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin. 相似文献
3.
Molecular clones of closed circular DNA molecules of a mink cell focus-inducing murine leukemia virus (MCF-13 MuLV) were generated. Closed circular DNA molecules isolated from a Hirt extraction of recently infected NIH/3T3 cells were inserted at their unique EcoRI site into lambda gtWES.lambda B. Restriction endonuclease analysis of inserts of two clones indicated that they represented intact MCF-13 MuLV genomes. One viral insert contained two large terminal repeat sequences, and the other contained only one. A 300-base-pair DNA fragment located in the envelope region of the MCF-13 MuLV genome was determined to be related to xenotropic MuLV sequences. 相似文献
4.
Virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type 1-infected cells. 总被引:27,自引:26,他引:1
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Monospecific antisera to herpes simplex virus type 1 (HSV-1) glycoproteins gB, gC, and gD were used to identify the HSV-1-specific glycoproteins associated with the nuclear fraction as compared with those associated with cytoplasmic fraction, whole-cell lysates, and purified virions. The results indicate that a predominance of HSV glycoprotein precursors pgC(105), pgB(110), and pgD(52) is associated with the nuclear fraction. Treatment of the nuclear fraction with the enzyme endo-beta-N-acetylglucosaminidase H indicated that the lower-molecular-weight glycoproteins are sensitive to this endoglycosidase. These results suggest that in the nuclear fraction of HSV-1-infected cells virus-specific glycoproteins gB, gC, and gD are predominately in the high-mannose precursor form; however, detectable amounts of the fully glycosylated forms of gC and gD were also found. 相似文献
5.
Expression of herpes simplex virus type 1 glycoproteins in interferon-treated human neuroblastoma cells. 总被引:1,自引:1,他引:0
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Human alpha interferon (IFN) significantly inhibits the replication of herpes simplex virus type 1 in human neuroblastoma cells. This inhibitory effect can be blocked by pretreatment with antiserum to IFN. We observed no significant differences in the expression of major nucleocapsid proteins, including VP5, between IFN-treated and untreated neuroblastoma cells. Electron micrographs demonstrated that there were distinct viral nucleocapsids within IFN-treated neuroblastoma cells. The expression of glycoproteins B and E was significantly reduced in these IFN-treated cells. On the other hand, glycoprotein D, although reduced in quantity, was expressed after IFN treatment. An immunofluorescence assay of the IFN-treated and virus-infected cells detected glycoprotein D in the Golgi complexes and in the nuclear membranes. Our results indicate that human alpha IFN may be useful in the study of gene expression in IFN-treated cells of neuronal origin. 相似文献
6.
N-acetylgalactosaminyltransferase activity involved in O-glycosylation of herpes simplex virus type 1 glycoproteins 总被引:3,自引:3,他引:0
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F Serafini-Cessi F Dall''Olio M Scannavini F Costanzo G Campadelli-Fiume 《Journal of virology》1983,48(1):325-329
We report on N-acetylgalactosaminyltransferase (UDPacetylgalactosamine--protein acetylgalactosaminyltransferase; EC 2.4.1.41) activity in herpes simplex virus type 1 (HSV-1)-infected BHK and RicR14 cells, a line of ricin-resistant BHK cells defective in N-acetylglucosaminyltransferase I. The enzyme catalyzed the transfer of [14C]N-acetylgalactosamine (GalNAc) from UDP-[14C]GalNAc into HSV glycoproteins, as identified by immunoprecipitation. The sugar was selectively incorporated into the immature forms of herpesvirus glycoproteins pgC, pgD, and gA-pgB, which are known to contain N-linked glycans of the high-mannose type. The high incorporation of [14C]GalNAc into endogenous acceptors of HSV-1-infected RicR14 cells was consistent with the accumulation of immature forms of HSV glycoproteins which occurs in these cells. Mild alkaline borohydride treatment of glycoproteins labeled via GalNAc transferase showed that the transferred GalNAc was O-linked and represented the first sugar added to the peptide backbone. 相似文献
7.
Synthesis and processing of glycoproteins gD and gC of herpes simplex virus type 1. 总被引:6,自引:42,他引:6
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Herpes simplex virus type 1 (HSV-1) contains five glycoproteins, designated gA, gB, gC, gD, and gE. The present studies focused on the synthesis and processing of two of these, gC and gD. By using monoprecipitin antibody to gC, we demonstrated an antigenic and structural relationship between the precursor, pgC(110), and the product, gC(130). Tryptic peptide analysis showed that pgC and gC shared methionine peptides and that these molecules had the same fingerprint pattern as that of gC(130) extracted from the purified virion. These results suggested that post-translational processing of gC involved no major changes in methionine-containing tryptic peptides or in the cleavage sites required to generate those peptides. The syntheses of gC and gD were compared. We found that the glycoproteins were synthesized starting at different times in the infectious cycle; pgD was detected by 2 h postinfection, whereas pgC was first detected at 4 to 6 h postinfection. Both precursor molecules, pgC(110) and pgD(52), are basic glycopolypeptides, and in both cases processing involved changes in molecular weight and charge. These changes were detected by two-dimensional gel electrophoresis. Both glycoproteins exhibited heterogeneity, displayed as a series of spots (6 for gD and 15 to 20 for gC) of increasing negative charge and molecular weight. Neuraminidase treatment decreased the size, number, and acidic charge of the spots, suggesting that processing was due in part, but not entirely, to addition of sialic acid to pgD and pgC. 相似文献
8.
Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2. 总被引:26,自引:21,他引:5
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N Balachandran D Harnish R A Killington S Bacchetti W E Rawls 《Journal of virology》1981,39(2):438-446
Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F. 相似文献
9.
Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1. 总被引:2,自引:0,他引:2
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The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases. 相似文献
10.
Endo-beta-N-acetylglucosaminidase H sensitivity of precursors to herpes simplex virus type 1 glycoproteins gB and gC. 总被引:32,自引:31,他引:1
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The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment. 相似文献
11.
12.
Galdiero S Falanga A Vitiello M Raiola L Fattorusso R Browne H Pedone C Isernia C Galdiero M 《The Journal of biological chemistry》2008,283(44):29993-30009
Glycoprotein H (gH) of herpes simplex virus type I (HSV-1) is involved in the complex mechanism of membrane fusion of the viral envelope with the host cell. Membrane interacting regions and potential fusion peptides have been identified in HSV-1 gH as well as glycoprotein B (gB). Because of the complex fusion mechanism of HSV-1, which requires four viral glycoproteins, and because there are only structural data for gB and glycoprotein D, many questions regarding the mechanism by which HSV-1 fuses its envelope with the host cell membrane remain unresolved. Previous studies have shown that peptides derived from certain regions of gH have the potential to interact with membranes, and based on these findings we have generated a set of peptides containing mutations in one of these domains, gH-(626-644), to investigate further the functional role of this region. Using a combination of biochemical, spectroscopic, and nuclear magnetic resonance techniques, we showed that the alpha-helical nature of this stretch of amino acids in gH is important for membrane interaction and that the aromatic residues, tryptophan and tyrosine, are critical for induction of fusion. 相似文献
13.
14.
Kelly BJ Diefenbach E Fraefel C Diefenbach RJ 《Biochemical and biophysical research communications》2012,417(3):961-965
The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein. 相似文献
15.
Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. 总被引:94,自引:75,他引:19
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The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis. 相似文献
16.
The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS. 相似文献
17.
Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells. 总被引:3,自引:1,他引:2
Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host. 相似文献
18.
Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC. 总被引:3,自引:1,他引:2
The kinetics of processing and transport of herpes simplex virus type 1 (HSV-1) glycoproteins gB and gC was investigated. The conversion of precursor to mature forms and the appearance of the glycoproteins at the infected-cell surface at different times postinfection (p.i.) were studied. gB, synthesized at 4 h p.i., was converted to the mature form with a half-time (t1/2) of 120 min and appeared at the plasma membrane with a t1/2 of 270 min. The gB synthesized at later times p.i. (6, 8, and 10.5 h) was transported less efficiently. Less than 50% of gB synthesized at later times p.i. was processed and transported to the cell surface. gB synthesized in transfected cells was transported to the plasma membrane with kinetics similar to that for gB synthesized at early times p.i. gC was processed efficiently when synthesized at both 8 and 10.5 h p.i., with t1/2 of conversion of pgC to gC of 40 and 60 min, respectively. Approximately 90 to 95% of the gC synthesized was converted to the mature form. The gC synthesized at 8 h p.i. was also transported rapidly to the cell surface, compared with the transport of gB synthesized at the same time, with a t1/2 of 240 min. Greater than 70% of the gC synthesized at 8 h p.i. appeared at the cell surface. The gC synthesized at 10.5 h was transported less efficiently to the cells surface during a 6-h chase. 相似文献
19.
Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants. 总被引:17,自引:13,他引:4
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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. 相似文献
20.