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)

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

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels > or = 70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P(60/62/64)) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. Increasing the amount of P(60/62/64) expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P(TTT)) had no apparent effect on transcription (113% of the wild-type level) or replication (81% of the wild-type level), substitution with phenylalanine (P(FFF)) rendered the protein much less active in transcription (< 5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P(EEE)) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P(RRR)) or transcription (P(60/62/64)) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L.
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PMID:Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. 934 67

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have recently shown that the P protein of VSV, New Jersey serotype (PNJ), expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present work, we are studying the role of phosphorylation in the activation of the P protein of Indiana serotype (PIND), which is highly nonhomologous in amino acid sequence yet structurally similar to its New Jersey counterpart. Despite the fact that E. coli-expressed PIND required phosphorylation by CKII for activation, the phosphorylation negative P protein mutants generated by altering the phosphate acceptors S and T to alanine, surprisingly, showed transcription activity similar to wild-type in vitro. Alteration of S and T residues to phenylalanine, similarly, supported substantial transcription activity (approx. 60% of wild-type), whereas substitution with arginine residue abrogated transcription (approx. 5% of wild-type). In contrast, the same mutants, when expressed in eucaryotic cells, exhibited greatly reduced transcription activity in vitro. This disparate display of transcription phenotype by the PIND mutants expressed in bacteria and eucaryotic cells suggests that these mutants are unique in assuming different secondary structure or conformation when synthesized in two different cellular milieu. The findings that, unless phosphorylated by CKII, the bacterially expressed unphosphorylated (P0) form of PIND, as well as the phosphorylation negative mutants expressed in eucaryotic cells, demonstrates transcription negative phenotype indicate that, like PNJ, phosphorylation of PIND is essential for its activity.
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PMID:Display of disparate transcription phenotype by the phosphorylation negative P protein mutants of vesicular stomatitis virus, Indiana serotype, expressed in E. coli and eucaryotic cells. 936 99

An apparently novel adenovirus was associated with an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California (USA) during 1993-1994. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. Six black-tailed yearling deer (O. hemionus columbianus) were inoculated with purified adenovirus isolated from a black-tailed fawn that died of acute adenovirus hemorrhagic disease during the epizootic. Three of six inoculated deer also received intramuscular injections of dexamethasone sodium phosphate every 3 days during the study. Eight days post-inoculation, one deer (without dexamethasone) developed bloody diarrhea and died. Necropsy and histopathologic findings were identical to lesions in free-ranging animals that died of the natural disease. Hemorrhagic enteropathy and pulmonary edema were the significant necropsy findings and there was microscopic vascular damage and endothelial intranuclear inclusion bodies in the vessels of the intestines and lungs. Adenovirus was identified in necrotic endothelial cells in the lungs by fluorescent antibody staining, immunohistochemistry and by transmission electron microscopy. Adenovirus was reisolated from tissues of the animal that died of experimental adenovirus hemorrhagic disease. Similar gross and microscopic lesions were absent in four of six adenovirus-inoculated deer and in the negative control animal which were necropsied at variable intervals during the 14 wk study. One deer was inoculated with purified adenovirus a second time, 12 wk after the first inoculation. Fifteen days after the second inoculation, this deer developed severe ulceration of the tongue, pharynx and rumen and necrotizing osteomyelitis of the mandible which was associated with vasculitis and thrombosis of adjacent large vessels and endothelial intranuclear inclusions. Transmission electron microscopy demonstrated adenovirus within the nuclei of vascular cells and immunohistochemistry demonstrated adenovirus antigen within tonsilar epithelium and in rare vessels.
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PMID:Experimental adenovirus hemorrhagic disease in yearling black-tailed deer. 939 65

Several investigators have recently examined the effect of Fas (CD95)-mediated apoptotic cell death on target cells (TC). The effect of Fas-mediated death on viral RNA within the TC, however, has not been explored. In this study, we investigated the ability of the Fas pathway to mediate pre-lytic degradation of vesicular stomatitis virus (VSV) RNA and TC RNA. We show that engagement of Fas antigen on VSV-infected Jurkat cells induces pre-lytic degradation of VSV RNA transcripts, whereas full-length VSV genome RNA, known to be tightly associated with viral proteins, is not degraded. Cellular RNA, including beta-actin and glyceraldehyde-3-phosphate-dehydrogenase mRNAs, is also degraded by Fas-mediated cytotoxicity. In addition, Fas-mediated cytotoxicity reduced the yield of VSV plaque-forming units (PFU) from Jurkat by an average of 82.0%. An anti-Fas blocking Ab inhibited the RNA degradation and restored the number of VSV PFU to near control levels. These data indicate that the Fas lytic pathway could play a role in the elimination of viruses through degradation of intracellular viral RNA. reserved
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PMID:Fas-mediated cytotoxicity induces degradation of vesicular stomatitis virus RNA transcripts and reduces viral titer. 951 64

We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.
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PMID:Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity. 966 Dec 60

The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3-5 min after treatment with 1 x 10(-7) M TRH, with dramatic reductions in plasma membrane localization achieved within 10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist.
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PMID:Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor. 972 16

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.
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PMID:Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels. 1002 67

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.
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PMID:Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P. 1036 10

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson-Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar-phosphate backbone of the RNA and leaves the Watson-Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.
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PMID:Structure of the RNA inside the vesicular stomatitis virus nucleocapsid. 1068 65


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