Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.
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PMID:Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. 215 55

Recombinant DNA techniques were used to delete regions of a cDNA clone of the phosphoprotein NS gene of vesicular stomatitis virus. The complete NS gene and four mutant genes containing internal or terminal deletions were inserted into a modified pGem4 vector under the transcriptional control of the phage T7 promoter. Run-off transcripts were synthesized and translated in vitro to provide [35S]methionine-labeled complete NS or deletion mutant NS proteins. Immune coprecipitation assays involving these proteins were developed to map the regions of the NS protein responsible for binding to the structural viral nucleocapsid protein N and the catalytic RNA polymerase protein L. The data indicate the NS protein is a bivalent protein consisting of two discrete functional domains. Contrary to previous suggestions, the negatively charged amino-terminal half of NS protein binds to L protein, while the carboxyl-terminal half of NS protein binds to both soluble recombinant nucleocapsid protein N and viral ribonucleocapsid template.
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PMID:Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus. 244 89

Thirteen distinct monoclonal antibodies to the 30-kDa NS phosphoprotein of vesicular stomatitis virus were isolated and assayed by Western blot analysis and immune precipitation reactions. Epitopes recognized by the antibodies were mapped by immune precipitation of NS deletion proteins synthesized in vitro from cloned NS gene constructs. None of the epitopes recognized by the 13 antibodies could be mapped to the phosphorylated amino-terminal half of the NS protein. Twelve of the antibodies recognized epitopes located within the carboxy-terminal 142 amino acids of the protein. The great majority of epitopes appeared to consist of a linear array of amino acids.
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PMID:Deletion mapping analyses indicate that epitopes for monoclonal antibodies to the NS phosphoprotein of VSV are linear and clustered. 245 13

The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-associated RNA polymerase L protein, transcribes the genome ribonucleoprotein template in vitro. It contains an acidic N-terminal domain and two distinct domains at the C-terminal end that are involved in binding to the polymerase protein and the template RNA enwrapped with the nucleocapsid protein. In the present study, the portions of the NS gene that encode the N- and C-terminal domains of the protein were cloned in pGEM vectors and expressed by in vitro transcription and translation. It was shown that two polypeptides obtained by translation of the encoded mRNAs support RNA synthesis in vitro in a reconstitution reaction when they are added together in trans. Moreover, the N-terminal domain can be functionally substituted by structurally similar polypeptides.
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PMID:Two separate domains within vesicular stomatitis virus phosphoprotein support transcription when added in trans. 282 61

Internal initiation of translation on the vesicular stomatitis virus (VSV) phosphoprotein (P) mRNA leads to the synthesis of a second protein [Herman, R. C. (1986) J. Virol. 58, 797-804]. Characterization of this phenomenon shows that initiation at the 5'-proximal and internal AUG codons has different optima for mono- and divalent cations in the reticulocyte lysate. Whereas 5' initiation is stimulated by increasing concentration of K+ over the endogenous level, internal initiation is inhibited. Internal initiation is much less sensitive to the effects of the cap analogue 7mGpppG in both the reticulocyte lysate and the wheat-germ extract under conditions that reduce 5'-proximal initiation to only about 4-5% of the control level. These results imply that 5'-proximal and internal initiations are distinct biochemical processes.
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PMID:Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA. 283 42

The interaction of the nucleocapsid protein N and the phosphoprotein NS of vesicular stomatitis virus (VSV) was studied, free of other viral proteins, by transcription from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. N-NS complex formation depended strongly on cotranslation of the two proteins; when N and NS were mixed following separate translation of each, very little complex formation occurred. Conditions were found under which at least six N-NS complexes were separated from each other by electrophoresis in a nondenaturing gel system, and the following findings were made. (i) These complexes fell into two groups; complexes 1 through 5 all had a stoichiometry of two molecules of N to one molecule of NS, whereas N-NS complex 6 had an equimolar ratio of the two proteins. (ii) N-NS complexes 1 through 5 predominated at lower concentrations of NS relative to N, but N-NS complex 6 was the major or sole product when NS was equimolar to or in excess of N. (iii) The two sets of complexes were formed by two distinct types of interactions of NS with N. The formation of N-NS complexes 1 through 5 was abolished by the removal of as few as 11 amino acid residues from the basic, highly conserved carboxy-terminal domain of NS, which is essential for the binding of NS to the N-RNA template of VSV. In contrast, formation of complex 6 was unaffected by removal of as many as 62 of the carboxy-terminal amino acids of NS, a region encompassing both the terminal basic domain and an adjacent domain which is required for VSV RNA polymerase function. The significance of these observations for the mechanism of VSV genome replication is discussed.
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PMID:Resolution of multiple complexes of phosphoprotein NS with nucleocapsid protein N of vesicular stomatitis virus. 283 92

The interactions between the nucleocapsid protein N and either RNA or the phosphoprotein NS of vesicular stomatitis virus (VSV) were studied by the transcription of N and NS mRNAs from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. Nascent N protein bound tightly to added labeled RNA, as well as to endogenous RNA in the reticulocyte lysate. This binding was demonstrated by three independent techniques. First, labeled N protein and labeled RNA migrated identically as a series of sharp, closely spaced bands in a nondenaturing gel system. Second, translated N protein behaved as a stable ribonucleoprotein complex in CsCl gradients and sedimented to the same density as the authentic N-RNA template of VSV. Third, translated N protein protected a series of labeled RNA fragments from digestion by RNase A. None of the three RNA-binding criteria was satisfied by either translated NS protein or two deletion mutants of N protein or by other components of the reticulocyte lysate. The evidence suggests that the observed binding of RNA by nascent N was not RNA sequence specific, in contrast to the encapsidation process during VSV replication. Moreover, the prior formation of N-NS complexes totally abolished the observed binding of RNA by N. Thus, we propose that NS may be responsible for conferring the sequence specificity of the RNA binding that occurs during VSV genome replication.
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PMID:Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. 283 93

A full-length cDNA clone of the mRNA encoding the phosphoprotein (NS) of the Indiana serotype of vesicular stomatitis virus was inserted into the SP6 transcription vector. By in vitro transcription of the inserted gene followed by translation of the mRNA in a rabbit reticulocyte lysate, NS protein was synthesized. The biological activity of the protein was demonstrated by RNA synthesis in vitro by reconstitution with L protein and N-RNA template purified from virions. Using oligonucleotide-directed RNase H cleavage of the full-length NS mRNA, a series of deleted RNAs were made which gave rise to corresponding size classes of truncated NS protein after translation in vitro. The N-RNA template binding site was located at the C-terminal domain (21 amino acids) of the NS protein and the L-protein binding site was present within 14 amino acids spanning the NH2-terminal side of the N-RNA binding site. These results are similar to that obtained with the NS protein of the New Jersey serotype of VSV, indicating conservation of the functional domains within the VSV serotypes.
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PMID:The functional domains of the phosphoprotein (NS) of vesicular stomatitis virus (Indiana serotype). 284 48

The phosphoprotein (NS) of vesicular stomatitis virus is an indispensable subunit of the virion-associated RNA polymerase (L). NS consists of a highly acidic NH2-terminal domain and a basic COOH-terminal domain. Unlike the latter, the amino acid sequences of the NH2-terminal regions are highly dissimilar among different viral serotypes, although they share structural similarities. We have cloned an NS gene into the SP6 transcription vector and replaced the 5'-terminal 80% by a full-length gene for beta-tubulin, which contains an acidic COOH-terminal domain. Here we present evidence that the chimeric tubulin-NS protein is biologically active and that the acidic region in tubulin directly affects the transcription reaction. These observations indicate that NS probably functions as an activator protein in which the acidic domain stimulates transcription of the viral genes by interacting with the RNA polymerase as observed for eukaryotic cellular transcription activators.
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PMID:NH2-terminal acidic region of the phosphoprotein of vesicular stomatitis virus can be functionally replaced by tubulin. 284 50

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


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