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1.
Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses 总被引:2,自引:1,他引:1
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Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. 相似文献
2.
A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein. 总被引:4,自引:3,他引:1
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In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does not promote intracellular transport directly but influences a polypeptide folding or oligomerization step which is critical for transport. 相似文献
3.
We have studied the effects of inhibiting the initial steps in processing of asparagine-linked oligosaccharides on the formation of vesicular stomatitis virus (VSV). Our data show that conditions which prevent the removal of glucose can block the growth of this virus. Our conclusion that inhibition of VSV synthesis is due specifically to an effect on the ability of the virus glycoprotein, G, to mature to a correct functional conformation is based on the following observations: (i) two drugs, deoxynojirimycin and castanospermine , both of which selectively inhibit the processing glucosidases, affected virus growth; (ii) only one of the two strains (San Juan and Orsay ) of VSV tested was affected and that strain, VSV(San Juan), is known to have a G protein highly sensitive to alterations in oligosaccharide structure; (iii) the effect was to make the formation of VSV(San Juan) temperature-sensitive, a result previously observed with alterations in the oligosaccharides on G protein; (iv) a cell variant missing glucosidase II activity also became temperature-sensitive in its ability to produce VSV(San Juan) but not VSV( Orsay ). Although inhibition of glucosidase activity by 1- deoxynojirimycin caused a 10-fold drop in virion formation, transport of G protein to the plasma membrane was not altered. The growth of VSV(San Juan) at 40 degrees C was not affected when subsequent steps in the processing pathway were blocked. These data indicate that by the time the glucose residues are removed G has attained a stable conformation. 相似文献
4.
Mutational changes in the vesicular stomatitis virus glycoprotein affect the requirement of carbohydrate in morphogenesis. 总被引:4,自引:4,他引:0
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The role of carbohydrate in the morphogenesis of vesicular stomatitis virus was studied, using the antibiotic tunicamycin to inhibit glycosylation. It has been reported previously (Gibson et al., J. Biol. Chem. 254:3600-3607, 1979) that the San Juan strain of vesicular stomatitis virus requires carbohydrate for efficient migration of the glycoprotein (G) to the cell surface and for virion formation, whereas the prototype or Orsay strain of vesicular stomatitis virus is less stringent in its carbohydrate requirement at 30 degrees C. However, there are many differences between the two strains. We found that mutational changes within the G protein of the same strain of virus (prototype or Orsay) alters the requirement for carbohydrate at 30 degrees C. Group V or G protein mutants tsO45 and tsO44, like their prototype parent, did not require carbohydrate for efficient morphogenesis. In contrast, the G protein of another group V mutant, tsO110, was totally dependent upon carbohydrate addition for migration to the cell surface. Furthermore, no tsO110 particles were released in the absence of glycosylation. The wild-type prototype strain did require carbohydrate at 39.5 degrees C for insertion of the G protein into the plasma membrane and virion formation. However, a pseudorevertant of tsO44 (tsO44R), unlike the prototype parent, no longer exhibited this temperature-sensitive requirement for carbohydrate. At 39.5 degrees C in the presence of tunicamycin, tsO44R-infected cells released normal yields of particles and the unglycosylated G reached the cell surface very efficiently. In contrast to tsO110, which absolutely requires carbohydrate, mutational change in the tsO44R G protein has eliminated the requirement for carbohydrate. Thus, simple mutational changes, as opposed to many changes in the molecule, are sufficient to alter the carbohydrate requirement. 相似文献
5.
Phosphorylation of avian retrovirus matrix protein by Ca2+/phospholipid-dependent protein kinase 总被引:4,自引:0,他引:4
The matrix protein from avian myeloblastosis virus and the Rous sarcoma virus, Prague C strain, is a phosphoprotein. A comparison of the amino acid sequences shows these phosphoproteins are very similar. The sites of phosphorylation of the matrix protein purified from virions are identified as serine residues 68 and 106. Treatment with purified rabbit skeletal-muscle protein phosphatase 1 or 2A, selectively releases phosphate from serine 68, while alkali treatment releases phosphate from both sites. When analyzed as a substrate for six different protein kinases, only the Ca2+/phospholipid-dependent protein kinase modifies the matrix protein. The serine residues phosphorylated in vivo are identical to those phosphorylated in vitro by this protein kinase. The role of these phosphorylation events in viral production is discussed. 相似文献
6.
7.
Phosphorylation of threonine in the proline-rich carboxy-terminal region of simian virus 40 large T antigen. 总被引:14,自引:13,他引:1
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The position of phosphothreonine in the predicted primary structure of simian virus 40 large T antigen was determined by different methods. After digestion of large T antigen with trypsin and subsequent two-dimensional peptide mapping, a single peptide containing phosphothreonine could be separated from the bulk of phosphoserine-containing peptides. Its amino acid composition was determined by differential labeling with various amino acids in vivo. The high yield of proline (4.5 mol) within the phosphothreonine peptide indicated that it was derived from the carboxy terminus of large T antigen and had in its unphosphorylated form the sequence Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr-COOH. A phosphopeptide generated by chymotrypsin could be converted into the tryptic phosphothreonine peptide, indicating that the latter was part of the chymotryptic peptide. The origin of the phosphothreonine-containing peptides was independently confirmed by using an antiserum directed against the carboxy terminus of large T antigen. This serum reacted specifically with the proline-rich, phosphothreonine-containing peptides. Further analysis by partial acid hydrolysis indicated that the internal threonine was phosphorylated. The unusual amino acid composition on both sides of the phosphothreonine and the possible function of this phosphorylation site are discussed. 相似文献
8.
A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. 总被引:32,自引:5,他引:27
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DNA sequences were determined for three cDNA clones encoding vesicular stomatitis virus glycoproteins from the tsO45 mutant (which encodes a glycoprotein that exhibits temperature-sensitive cell-surface transport), the wild-type parent strain, and a spontaneous revertant of tsO45. The DNA sequence analysis showed that as many as three amino acid changes could be responsible for the transport defect. By recombining the cDNA clones in vitro and expressing the recombinants in COS cells, we were able to trace the critical lesion in tsO45 to a single substitution of a polar amino acid (serine) for a hydrophobic amino acid (phenylalanine) in a hydrophobic domain. We suggest that this nonconservative substitution may block protein transport by causing protein denaturation at the nonpermissive temperature. Comparison of the predicted glycoprotein sequences from two vesicular stomatitis virus strains suggests a possible basis for the differential carbohydrate requirement in transport of the two glycoproteins. 相似文献
9.
Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation. 总被引:22,自引:18,他引:4
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The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine. 相似文献
10.
Envelope Proteins of Vesicular Stomatitis Virus: Effect of Temperature-Sensitive Mutations in Complementation Groups III and V 总被引:57,自引:30,他引:27
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Florence Lafay 《Journal of virology》1974,14(5):1220-1228
All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations. 相似文献
11.
12.
13.
S Fischer A Flamand R Fagard J F Chich J P Piau L Reibel G Gacon 《The Journal of biological chemistry》1985,260(27):14406-14409
Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56. 相似文献
14.
Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein. 总被引:5,自引:0,他引:5
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The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo. 相似文献
15.
16.
Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions. 总被引:2,自引:16,他引:2
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Vesicular stomatitis virus was extracted with 60 mM octylglucoside in the absence of salts and in the presence of 0.5 M NaCl. The resulting extracted virus particles were examined by electron microscopy, and the proteins present were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extraction in the absence of salts yielded subviral structures which we cell "skeletons" as originally suggested by Cartwright et al. (J. Gen. Virol. 7:19-32, 1970). The skeletons contained the viral N, M, and L proteins, but they lacked the glycoprotein (G) entirely. Morphologically, the skeletons resembled intact vesicular stomatitis virus but they were slightly longer and smaller in diameter. Like native vesicular stomatitis virus, skeletons were found to have lateral striations spaced 5.0 to 6.0 nm apart along the length of the structure. In contrast to extraction in the absence of NaCl, extraction of vesicular stomatitis virus with 60 mM octylglucoside in the presence of 0.5 M NaCl yielded highly extended viral nucleocapsids in which N was the predominant protein; no M or G proteins could be detected. These results support the view that the M protein is involved in maintaining the nucleocapsid in the compact form found in native virions. 相似文献
17.
Phosphorylation pattern of large T antigens in mouse cells infected by simian virus 40 wild type or deletion mutants 总被引:8,自引:6,他引:2
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The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been extensively studied in productive infection of monkey cells. In this study, we analyzed the phosphorylation sites of large T antigen from SV40-infected nonpermissive mouse cells by partial proteolysis fingerprints and analysis of the phosphoamino acids present in the resulting fragments. The wild-type virus and deletion mutants (dl1263, dl1265, dl2194, and dl2198) were used for infection. On the basis of our results and published data (M. Schwyzer, R. Weil, and H. Zuber, J. Biol. Chem. 225:5627-5634, 1980), a cleavage map of large T antigen was established. It was reported that at least four sites of phosphorylation were present. The amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the prolinerich C-terminal end of the molecule at position 701 or 708. On the basis of the concensus as to the amino acid sequence surrounding the recognition sites for protein kinases, it was possible to more precisely locate this phosphothreonine at residue 701. Moreover, the C-terminal part of the molecule contained phosphoserine at a more internal position. In addition, this study firmly established the presence of a phosphothreonine in the N-terminal part of large T antigen. In conclusion, it was shown that the location of phosphorylation sites of large T antigen produced by nonpermissive mouse cells infected by SV40 is strikingly similar to that reported by other groups for large T antigen produced by SV40-infected permissive cells. 相似文献
18.
Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.
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Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface. 相似文献
19.
Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo 总被引:9,自引:0,他引:9
The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function. 相似文献
20.
Localization of the membrane-associated region of vesicular stomatitis virus M protein at the N terminus, using the hydrophobic, photoreactive probe 125I-TID. 总被引:10,自引:9,他引:1
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The membrane-reactive, photoactivatable probe 125I-TID [3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-3H-diazirine] was found to label the M protein of vesicular stomatitis virus about 40% as much as G protein in intact virions, in agreement with labeling studies with other probes. By analyzing limited tryptic digestion and specific chemical cleavage products, the label was essentially entirely localized within the first 19, and probably within the first 5 to 10, amino acid residues at the N terminus, identifying this short amphipathic segment as the likely site of interaction of M protein with the viral bilayer. 相似文献