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)

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.
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PMID:Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase. 838 99

The sequence of 5568 nucleotides of the 3' moiety of the Mokola virus genome (serotype 3 of lyssaviruses) encompassing the nucleoprotein (N), phosphoprotein, matrix protein, and glycoprotein genes is presented and compared to that of the vaccinal strains of serotype 1. It allowed us to determine consensus sequences derived from the transcriptional start/stop signals and the order of protein conservation (nucleoprotein > matrix protein > phosphoprotein) in lyssaviruses. The sequences of the N gene of a fox rabies virus isolate from France (serotype 1), Lagos bat virus (serotype 2), Duvenhage virus (serotype 4), two European bat lyssaviruses (EBL) subtype 1, and two EBL subtype 2 were also determined to study the genetic diversity throughout the whole Lyssavirus genus and reinvestigate the classification of this genus. Six clearly distinct genotypes can be distinguished according to their percentage of amino acid similarity. Genotypes 2 (Lagos bat virus) and 3 (Mokola virus) are the most phylogenetically distant from the vaccinal and classical rabies viruses of genotype 1. Genotypes 4 (Duvenhage virus) and 5 (EBL1) are closely related to each other. Genotype 6 is represented by EBL2. Compared to the N proteins of the four principal serotypes of the Vesiculovirus genus (vesicular stomatitis virus serotype New Jersey and serotype Indiana, Chandipura virus, and Piry virus), the N gene of lyssaviruses exhibits a lower genetic variability.
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PMID:Molecular diversity of the Lyssavirus genus. 838 91

Interferon-beta (IFN-beta) strongly inhibited the expression of the hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), a paramyxovirus, in HeLa cells under the conditions where it did not affect the expression of the four upstream genes encoding the nucleocapsid protein, phosphoprotein, membrane protein and fusion protein. Even the downstream gene, encoding the large protein as well as the genome replication, appeared to be less susceptible to IFN-beta than the HN gene. This selective action of IFN-beta did not appear to be attributable to its well characterized antiviral mechanisms such as acceleration of RNA decay and translation inhibition. No similar down-regulation of a particular gene expression was found with another paramyxovirus, Sendai virus, or with a rhabdovirus, vesicular stomatitis virus, or seems to have been reported previously with any negative-strand RNA viruses. This new effect of IFN-beta thus suggests gene expression mechanism unique to NDV and may further lead to the discovery of a novel biochemical effect of IFN-beta.
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PMID:Antiviral action of interferon-beta on Newcastle disease virus: selectivity to the hemagglutinin-neuraminidase gene expression. 853 78

The growth of vesicular stomatitis virus requires two distinct RNA synthetic events: transcription of messenger RNA molecules and replication of the viral genome RNA. We report the use of a panel of monoclonal antibodies directed against the viral phosphoprotein P in an attempt to assess the role of this protein in RNA synthesis. Using extracts derived from virus-infected cells, we show that several anti-P monoclonal antibodies can have an inhibitory effect on genome RNA replication by binding to a soluble form of the P protein. We also show that the P protein to which one of these antibodies (6D11) is directed is not complexed with the N protein and that the amount of soluble P protein that binds to the 6D11 antibody in immunoprecipitation reactions can be increased by treating extracts with alkaline phosphatase. In addition, phosphatase treatment of infected cell extracts results in an increased level of genome RNA replication. These results suggest that a soluble subspecies of the P protein that functions in genome RNA replication exists in infected cells and that this species of the P protein is not required for transcription.
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PMID:Inhibition of VSV genome RNA replication but not transcription by monoclonal antibodies specific for the viral P protein. 861

By sequencing the 3 half of the Piry virus genome, we show that Piry virus, like the other vesiculoviruses, contains the genes for nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G and polymerase protein L, in that order. Our analysis of the Piry G protein sequence suggests that Piry and Chandipura are related to each other as closely as the Indiana and New Jersey vesicular stomatitis virus serotypes are to each other. A re-examination of amino acid sequences in the nucleocapsid protein shows that this relationship is also true of the more conserved central region of this protein and that the greatest divergence between Piry and Chandipura has occurred in two other regions of the nucleocapsid protein.
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PMID:The relationship of Piry virus to other vesiculoviruses: a re-evaluation based on the glycoprotein gene sequence. 872 58

Overexpression of a clone of vesicular stomatitis virus phosphoprotein P (New Jersey serotype) using T7 promoter with phoA leader sequence and a simpler two-step purification procedure of the expressed protein has been developed. The purified protein retains its ability to activate the transcription reaction. Comparative transcriptional assay using the protein P purified from periplasmic space and from cytosol (in the form of inclusion body) of Escherichia coli establishes the fact that the former is 10 times more efficient than the latter in activating the transcription reaction in vitro.
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PMID:Periplasmic expression of biologically active vesicular stomatitis virus phosphoprotein P in Escherichia coli. 877 56

The phosphoprotein (P) of vesicular stomatitis virus was previously shown to assemble into a homomultimer upon phosphorylation by casein kinase II. It thus acquired transcriptional activity, including the ability to bind to the other two transcriptional components, the polymerase L and the N-RNA template. This multimer has now been found to be a trimer using a His-tag dilution method. Trimer stability was assessed using a variation of this method, by measuring the rate of exchange of monomers between preformed tagged and untagged trimers at different values of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer was completely dissociated at pH 4. This suggests that the trimer is stabilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P trimers, completely preventing subunit exchange under transcription conditions. The association constants (Kass) for trimerization of partially active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, further confirming that the active species is the trimer. Circular dichroism spectra were identical for phosphorylated and unphosphorylated wild-type P protein and for D and A mutants known to be predominantly trimeric and monomeric, respectively.
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PMID:The transcriptional form of the phosphoprotein of vesicular stomatitis virus is a trimer: structure and stability. 893 54

We have previously shown that the phosphoprotein (P) of vesicular stomatitis virus (VSV), New Jersey serotype (PNJ) is phosphorylated by casein kinase II, within the N-terminal domain I (P1 form), whereas the C-terminal domain II is phosphorylated by a protein kinase activity associated with the L protein (P2 form) (D. J. Chattopadhyay and A.K. Banerjee, Cell 49, 407, 1987; A.M. Takacs et al., J. Virol. 66, 5842, 1992). In the present studies, we have mapped the corresponding P1 and P2 phosphorylation sites in the P protein of the well-studied Indiana serotype (PIND) and compared that with the two previously designated NS1 and NS2 forms present in vivo. The PIND expressed in Escherichia coli in an unphosphorylated form (P0) was used as substrate for recombinant casein kinase II (CKII). By site-directed mutagenesis, the CKII-mediated phosphorylation sites in the P protein were mapped at S60, T62, and S64 within the acidic domain I in vitro. In contrast, using BHK cell extract as the source of CKII or expressing P protein in COS cells labeled with 32PI, the phosphorylation sites were mapped at S60 and S64 with no phosphorylation at T62 residue. We used a peptide mapping technique by which the phosphorylation sites within domain I and domain II were determined. Using this method we demonstrated that the P1 and P2 forms are similar, if not identical, to the previously designated NS1 and NS2 forms, respectively. The domain II phosphorylating kinase activity, associated with the L protein, is shown to be present also in the N-RNA complex, indicating that this activity is of cellular origin. By site-directed mutagenesis, we have shown that S226 and S227 are involved in phosphorylation within domain II. We also demonstrate that the P1 and P2 forms are interconvertible and arise by phosphorylation/dephosphorylation of the phosphate groups in domain II, confirming the precursor-product relationship between the two phosphorylated forms of P protein.
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PMID:Phosphorylated states of vesicular stomatitis virus P protein in vitro and in vivo. 912 26

The nucleocapsid protein (N) and phosphoprotein (P) genes of vesicular stomatitis virus (VSV), Indiana serotype, were coexpressed in Escherichia coli BL21(DE3) by using the expression vector pET-3a. The coexpression resulted in the formation of N-P complex. The purified N-P complex was found to inhibit transcription in vitro mediated by viral ribonucleoprotein (RNP) complex in a dose-dependent manner. However, addition of uninfected mammalian cell extracts together with the N-P complex to the transcribing RNP resulted in the synthesis of full-length negative-strand genome RNA. These results indicate that the N-P complex regulated transcription and a cellular factor(s) in combination with the N-P complex may switch the RNA polymerase from transcription to replication mode.
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PMID:Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro. 915 13

The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.
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PMID:Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger. 921 92


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