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1.
The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.  相似文献   

2.
Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus   总被引:13,自引:10,他引:3       下载免费PDF全文
Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [3H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [3H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)3(GlcNAc)2 peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [3H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [35S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.  相似文献   

3.
Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.  相似文献   

4.
Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.  相似文献   

5.
In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.  相似文献   

6.
Matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) spectrometry is a recently developed soft ionisation mass spectrometry technique which appears as a highly efficient tool for the N-glycosylation analysis of glycoproteins. The potentiality of this analytical technique is illustrated through the analysis of the N-glycosylation of the isolectin L of bean phytohemagglutinin (PHA-L). The analysis was carried out on the native PHA-L as well as on the N-glycans released from this lectin. Furthermore, the two glycopeptides containing the potential N-glycosylation sites prepared by proteolytic cleavage of PHA-L and purified by HPLC were analysed by MALDI-TOF. This study has confirmed that PHA-L is N-glycosylated by two populations of oligosaccharides, high-mannose-type N-glycans and paucimannosidic-type N-glycans, located on Asn-12 and Asn-60, respectively, and has pointed out the microheterogeneity of the glycans N-linked on both Asn residues.  相似文献   

7.
After treatment with swainsonine, an inhibitor of both lysosomal alpha-mannosidase and Golgi alpha-mannosidase-II activities, analysis of [3H]mannose-labeled glycans showed that HT-29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N-glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT-29 cells hybrid-type chains were detected, whereas undifferentiated HT-29 cells accumulated high-mannose-type oligosaccharide, despite our demonstration of Golgi alpha-mannosidase-II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannose-type chains in undifferentiated HT-29 cells was the result of the stabilization of glycoproteins substituted with these glycans. These data suggest that in undifferentiated HT-29 cells, glycoproteins with high-mannose-type oligosaccharides are delivered to a degradative compartment containing swainsonine-sensitive alpha-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which alpha-mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N-glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.  相似文献   

8.
Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.  相似文献   

9.
Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.  相似文献   

10.
The biological activity of two glycoproteins, hemagglutinin and neuraminidase (HN) and fusion (F) proteins, of Sendai virus (HVJ) were studied using purified proteins. The proteins were purified by chromatography on DEAE and CM cellulose in the presence of Nonidet P-40 (NP40). The glycoproteins were reconstituted at various ratios of F to HN into lipid vesicles containing fragment A of diphtheria toxin. The association of HN and F proteins with the vesicles was confirmed by electron microscopy and sucrose density gradient centrifugation. The cytotoxic activity of vesicles containing fragment A on fusion with L cells was determined by measuring colony formation of the cells. It was found that for maximum cytotoxic activity of the vesicles, there was an optimal ratio of F to HN of two. This suggests that HN is not merely the initial binding site to the cell surface, and that interactions between HN and F proteins on the virus surface may be important for the biological activities of these proteins on the cells.  相似文献   

11.
Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.  相似文献   

12.
Based on subcellular fractionation data, the following maturation pathways were proposed for the Newcastle disease virus glycoproteins. During or shortly after synthesis in rough endoplasmic reticulum, hemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins underwent dolichol pyrophosphate-mediated glycosylation, and HN assumed a partially trypsin-resistant conformation. HN began to associate into disulfide-linked dimers in rough endoplasmic reticulum, and at least one of its oligosaccharide side chains was processed to a complex form en route to the cell surface. During migration in intracellular membranes, F0 was proteolytically cleaved to F1.2. Neither HN nor F1,2 required oligosaccharide side chains for migration to plasma membranes, and cleavage of F0 also occurred without glycosylation. Virion- and plasma membrane-associated HN contained both complex and high-mannose oligosaccharide chains on the same molecule, and F1,2 contained at least high-mannose forms. Several of the properties of HN were notable for a viral glycoprotein. The oligosaccharide side chains of HN were modified very slowly in chick cells, whereas those of the G glycoprotein of vesicular stomatitis virus were rapidly processed to a complex form. Therefore, their different rates of migration and carbohydrate processing were intrinsic properties of these glycoproteins. Consistent with its slow maturation, the HN glycopolypeptide accumulated to high levels in intracellular membranes as well as in plasma membranes. Intracellular HN contained immature oligosaccharide side chains, suggesting that it accumulated in the pre-Golgi/Golgi segment of the maturation pathway. The major site of accumulation of mature HN with neuraminidase activity was the plasma membrane.  相似文献   

13.
This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.  相似文献   

14.
A novel chemoenzymatic approach to synthesize neoglycoproteins containing high-mannose-type oligosaccharides is described. p-Isothiocyanatophenyl-beta-d-glucopyranoside (Glc-ITC) was transferred to the reducing end of the high-mannose-type oligosaccharides using a transglycosylation activity of endo-beta-N-acetylglucosaminidase A (Endo-A). A novel oligosaccharide, Man(6)GlcNAc-Glc-ITC, was synthesized as a coupling reagent for lysyl and N-terminal residues of the protein moiety. The neoglycoconjugate was coupled with several nonglycosylated proteins such as ribonuclease A, lysozyme, and alpha-lactalbumin. Between one and four high-mannose-type oligosaccharides were incorporated per molecule of these proteins. This method should be very useful for the synthesis of neoglycoproteins with homogeneous high-mannose-type oligosaccharides.  相似文献   

15.
In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.  相似文献   

16.
The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.  相似文献   

17.
S Bagai  R A Lamb 《Journal of virology》1995,69(11):6712-6719
To compare the requirements for paramyxovirus-mediated cell fusion, the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of simian virus 5 (SV5), human parainfluenza virus 3 (HPIV-3), and Newcastle disease virus (NDV) were expressed individually or coexpressed in either homologous or heterologous combinations in CV-1 or HeLa-T4 cells, using the vaccinia virus-T7 polymerase transient expression system. The contribution of individual glycoproteins in virus-induced membrane fusion was examined by using a quantitative assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecyl rhodamine (R18) and a quantitative assay for content mixing based on the cytoplasmic activation of a reporter gene, beta-galactosidase. In these assays, expression of the individual F glycoproteins did not induce significant levels of cell fusion and no cell fusion was observed in experiments when cells individually expressing homologous F or HN proteins were mixed. However, coexpression of homologous F and HN glycoproteins resulted in extensive cell fusion. The kinetics of fusion were found to be very similar for all three paramyxoviruses studied. With NDV and HPIV-3, no cell fusion was detected when F proteins were coexpressed with heterologous HN proteins or influenza virus hemagglutinin (HA). In contrast, SV5 F protein exhibited a considerable degree of fusion activity when coexpressed with either NDV or HPIV-3 HN or with influenza virus HA, although the kinetics of fusion were two- to threefold higher when the homologous SV5 F and HN proteins were coexpressed. Thus, these data indicate that among the paramyxoviruses tested, SV5 has different requirements for cell fusion.  相似文献   

18.
Incubation of herpes simplex virus type 1-infected Vero and HEp-2 cells at a reduced temperature (34 degrees C) enhanced the detection of the nonglycosylated precursors (pgB97 and pgC75) to the gB and gC glycoproteins in the cytoplasmic and nuclear fractions. Relative to the fully glycosylated and high-mannose forms detected, the nonglycosylated precursors were the predominant components associated with the nuclear fraction of infected cells. Furthermore, addition of protease inhibitors to the fractionation buffers did not affect the distribution or abundance of the nonglycosylated precursors, suggesting that the presence of pgB97 and pgC75 was not the result of proteolysis. When infected Vero or HEp-2 cells were harvested at various times postinfection, the nonglycosylated precursors were detected after the initial appearance of the high mannose components (pgB110 and pgC105). In Vero cells, pgB97 and pgC75 were detected simultaneously at 8 h postinfection, whereas detection was not apparent in HEp-2 cells until 20 h postinfection. Conditions which favored detection of appreciable amounts of nonglycosylated precursors provided an unique approach to probe possible post-translational modifications in the absence of inhibitors of glycosylation. In nuclear fractions isolated from cycloheximide-treated HEp-2 or Vero cells, numerous discrete gC-immunoreactive bands migrating with decreased electrophoretic mobility relative to the nonglycosylated precursor pgC75 were observed. This series of one to four additional bands was eliminated by digestion with endoglycosidase H, and the appearance of these bands was blocked by the addition of tunicamycin. Collectively, the data suggest that high-mannose core oligosaccharides may be added to the nonglycosylated precursor of the gC glycoprotein of herpes simplex virus type 1 in a post-translational fashion.  相似文献   

19.
Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.  相似文献   

20.
Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.  相似文献   

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