Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription-competent cores of vesicular stomatitis virus (VSV) contain two tightly bound protein kinase activities capable of phosphorylating the viral P protein (Beckes and Perrault, Virology 184, 383-386, 1991). We examined here the specificity of these kinases for the P protein substrate and their activity during the in vitro transcription process. Conditions favoring the VSVK1 kinase activity resulted in phosphorylation of the P1 species predominantly whereas conditions favoring VSVK2, or transcription conditions, led to an increase in the proportion of the faster migrating P2 and P3 species. A minimum of 2 mol phosphate/mol P protein was incorporated in 1 hr under optimal transcription conditions. Pulse-chase experiments revealed that the VSVK2 activity converted phosphorylated P1 to P2/P3 species. Most or all of the sites modified by VSVK1 (serines only) mapped to the 78 amino acid-long N-terminal fragment of the P protein; additional serine acceptor sites of undetermined location were also phosphorylated under VSVK2 conditions. Pretreatment of virion cores with 5'-p-fluorosulfonylbenzoyl adenosine had little or no effect on P1 phosphorylation but inhibited P1 to P2/P3 conversion nearly completely, with no effect on subsequent transcription. Likewise, the addition of cell extracts had relatively little effect on P1 phosphorylation but strongly inhibited the appearance of P2/P3, without affecting concurrent transcription. We conclude that phosphorylation of the P protein during transcription in vitro is a two-step process carried out by two distinct kinase activities, but only the first step may be essential for viral mRNA synthesis.
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PMID:Stepwise phosphorylation of vesicular stomatitis virus P protein by virion-associated kinases and uncoupling of second step from in vitro transcription. 131 76

We mapped the in vivo phosphorylation sites for the matrix (M) protein of the Orsay and San Juan strains of vesicular stomatitis virus, Indiana serotype, using limited proteolysis and phosphoamino acid analysis. M protein was solubilized from 32P-labeled virions by using detergent and high-salt conditions, then treated with either trypsin or Staphylococcus aureus V8 protease, and analyzed by polyacrylamide gel electrophoresis and autoradiography to determine which fragments contained phosphate residues. The M protein fragment extending from amino acid 20 to the carboxy terminus contained approximately 70% of the control 32P label, while the fragment extending from amino acid 35 to the carboxy terminus had only trace amounts of label. These data indicate that the major phosphorylation site was between amino acids 20 and 34 in the Orsay strain M protein. Phosphoamino acid analysis of M protein by thin-layer electrophoresis showed the presence of phosphothreonine and phosphoserine and that phosphothreonine continued to be released after prolonged vapor-phase acid hydrolysis. These data identify Thr-31 as the primary in vivo phosphate acceptor for M protein of the Orsay strain of vesicular stomatitis virus. The San Juan strain M protein has serine at position 32, which may also be an important phosphate acceptor. In addition, phosphorylation at Ser-2, -3, or -17 occurs to a greater extent in the San Juan strain M protein than in the Orsay strain M protein. The subcellular distribution of phosphorylated M protein was investigated to determine a probable intracellular site(s) of phosphorylation. Phosphorylated M protein was associated primarily with cellular membranes, suggesting phosphorylation by a membrane-associated kinase. Virion M protein was phosphorylated to a greater extent than membrane-bound M protein, indicating that M protein phosphorylation occurs at a late stage in virus assembly. Phosphorylation of wild-type and temperature-sensitive mutant M protein was studied in vivo at the nonpermissive temperature. The data show that phosphorylated M protein was detected only in wild-type virus-infected cells and virions, suggesting that association with nucleocapsids may be required for M protein phosphorylation or that misfolding of mutant M protein at the nonpermissive temperature prevents phosphorylation.
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PMID:Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein. 132 2

The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphoprotein and the crucial role played by a cell protein kinase in this process.
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PMID:Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. 132 45

The vesicular stomatitis virus (VSV) NS and M proteins are not only phosphorylated in vivo but are also further modified by the virion-associated protein kinase(s) concomitantly with the in vitro transcription process. Although NS phosphorylation is necessary for this transcription, no function has yet been ascribed for M protein phosphorylation. We show here that all phosphates added to M protein in vitro mapped to the trypsin-sensitive N-terminal basic domain (residues 1-43). The major site(s) (approximately 93%) corresponded to one or more of three serine residues within the first 17 amino acids. Nearly 1 mol phosphate/mol protein was added in vitro under optimal conditions suggesting that only one of these three candidate serine residues corresponds to the major site. This same M protein domain is thought to play an important role in virus RNA synthesis by inhibiting transcription. We show here that in vitro phosphorylation did not appear to affect this function. Two critical serine residues in the VSV NS protein were previously reported to be phosphorylated during in vitro transcription (D. Chattopadhyay and A. K. Banerjee, 1987, Cell 49, 407-414). The sequence flanking these NS serines is very acidic while that of all three candidate phosphoserines in the M protein is very basic. We therefore predict that at least two distinct serine-specific kinase activities are packaged in virions, one specific for M and one specific for NS.
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PMID:Phosphorylation of vesicular stomatitis virus M protein: evidence for a second virion-associated protein serine kinase activity. 253 29

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.
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PMID:A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. 298 3

The phosphoprotein NS of vesicular stomatitis virus which accumulates within the infected cell cytoplasm is phosphorylated at multiple serine and threonine residues (G. M. Clinton and A. S. Huang, Virology 108:510-514, 1981; Hsu et al., J. Virol. 43:104-112, 1982). Using incomplete chemical cleavage at tryptophan residues, we mapped the major phosphorylation sites to the amino-terminal half of the protein. Analysis of phosphate-labeled tryptic peptides suggests that essentially all of the label is within the large trypsin-resistant fragment predicted from the sequence of Gallione et al. (J. Virol. 39:52-529, 1981). A similar result has been obtained for NS protein isolated from the virus particle by C.-H. Hsu and D. W. Kingsbury (J. Biol. Chem., in press). Analysis of phosphodipeptides utilizing the procedures of C. E. Jones and M. O. J. Olson (Int. J. Pept. Protein Res. 16:135-142, 1980) enabled us to detect as many as six distinct phosphate-containing dipeptides. From these studies, together with the known sequence data, we conclude that the major phosphate residues on cytoplasmic NS protein are located in the amino third of the NS molecule and most probably between residues 35 and 106, inclusive. The studies also provide formal chemical proof that NS protein has a structure consistent with a monomer of the sequence of Gallione et al. as modified by J. K. Rose (personal communication). The low electrophoretic mobility of this protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not therefore due to dimerization.
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PMID:Phosphorylation sites on phosphoprotein NS of vesicular stomatitis virus. 298 24

The complete nucleotide sequence of the NS mRNA of vesicular stomatitis virus (New Jersey serotype) was established from two cDNA clones spanning the entire coding region of the mRNA. The gene is 856 nucleotides long and can code for a polypeptide of 274 amino acids. Comparison with the nucleotide sequence of the NS gene of the Indiana serotype revealed only 41% sequence homology. The deduced amino acid sequences of the NS proteins were only 32% homologous, with no identical stretches of more than five amino acids. However, at the C-terminal domain there was a conserved region of 21 amino acids with greater than 90% homology. Surprisingly, relative hydropathicity plots also demonstrated the presence of a large number of hydrophilic amino acids sequestered similarly over the N-terminal half of the protein. In addition, the total number of serine and threonine residues, presumptive phosphorylation sites, was similar and included seven serine and three threonine residues located at identical positions. It appears that during divergent evolution of these two vesicular stomatitis virus serotypes from a common ancestor, considerable mutation occurred in the main body of the gene but the overall structure of the protein was retained. The function of the NS protein in relation to the evolution of the two viruses is discussed.
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PMID:Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology. 298 60

The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication.
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PMID:Cloning and expression of a viral phosphoprotein: structure suggests vesicular stomatitis virus NS may function by mimicking an RNA template. 301 52

Saccharomyces cerevisiae strains transformed with plasmids containing cDNAs coding for the glycoproteins of vesicular stomatitis or Sindbis viruses can be induced to produce large amounts of glycosylated virus glycoproteins. Studies reported here show that these proteins from high molecular weight disulfide-linked oligomers in the yeast endoplasmic reticulum. Oligomers were also found for two genetically altered forms of VSV G; one of these was lacking the membrane anchor domain and the other had the cysteine in the cytoplasmic tail replaced with serine. These oligomers can be separated from the bulk of yeast proteins by brief high-speed centrifugation of yeast extracts prepared by boiling cells with 1% sodium dodecyl sulfate. Treatment with thiol-reducing agents converts the oligomers to soluble monomeric forms, and this procedure leads to a substantial purification of glycoproteins from bulk yeast protein.
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PMID:Expression of genes encoding vesicular stomatitis and Sindbis virus glycoproteins in yeast leads to formation of disulfide-linked oligomers. 301 85

The nucleotide sequence of the 3' end of the genome of Chandipura (CHP) virus, including the complete sequences of the nucleocapsid (N) and phosphoprotein (NS) genes was determined, principally from cloned cDNAs of the N and NS mRNAs. The NS mRNA of CHP virus is 908 bases in length and encodes a protein of 293 amino acids. Comparison of the CHP virus NS protein sequence with those of vesicular stomatitis virus of the New Jersey serotype (VSV (NJ)) and of the Indiana serotype (VSV (IND] revealed homologies of only 23 and 21%, respectively, with no consecutive stretches of more than four amino acids identical among the three sequences. As with the two VSV serotypes, the highest homology between the NS proteins of CHP and VSV was in a 20-amino acid region near the carboxy termini of the proteins. Of the potential phosphorylation sites, there are eight conserved serine or threonine residues among the three sequences. Despite the dissimilarity among primary sequences of the NS proteins, their overall structure, as assessed by amino acid composition and by the relative hydropathicities of the sequences, has been conserved throughout evolution. The N mRNA of CHP virus is 1291 bases long and encodes a protein of 422 amino acids. In contrast to the NS protein, the CHP N protein is at least 50% homologous to the N proteins of each of the VSV serotypes. We have identified a region near the center of these N protein sequences which is conserved among members of both the rhabdovirus and paramyxovirus families. This extent of conservation of the N protein sequences underscores the high rate of mutability of the NS protein sequences among the vesiculoviruses.
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PMID:Sequences of Chandipura virus N and NS genes: evidence for high mutability of the NS gene within vesiculoviruses. 302 73


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