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)

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

Two vesicular stomatitis virion proteins, NS and M, are phosphorylated in vivo before packaging and in vitro during the transcription process carried out with disrupted virions. Phosphorylation of NS is thought to be essential for transcription but which of the many acceptor sites is or are involved in this function and which protein kinase(s) is responsible still need to be resolved. We recently reported that the virion-associated kinase which modifies M protein is most likely a different enzyme than that phosphorylating NS (Beckes et al., Virology 169, 161-171 1989). Here we present additional evidence for the presence of distinct enzymes modifying M vs NS substrates and also show that at least two distinct kinase activities modify NS protein. Each of the three activities displayed different optimum reaction conditions, phosphate donor preferences, and sensitivity to inhibition by N-ethylmaleimide.
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PMID:Two distinct protein kinase activities in vesicular stomatitis virions phosphorylate the NS transcription factor. 165 98

The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
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PMID:Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. 216 40

Constitutive expression of the type I interferon-inducible human cytoplasmic MxA protein has been shown to interfere with primary transcription of vesicular stomatitis virus (VSV) in tissue culture cells. As phosphorylation of the VSV P protein has been linked to its ability to stimulate viral transcription, we analyzed the phosphorylation status of this protein in human brain cells (U-87) stably transfected with MxA. We observed a general increase in cellular kinase activity in the presence of MxA, affecting both cellular proteins and VSV P protein. Phosphorylation of the latter was up to threefold higher both in vivo and in vitro. In vitro phosphorylation of recombinant VSV P protein could be enhanced in MxA-negative cell extracts after exogenous addition of recombinant His-MxA. Biochemical evidence and phosphorylation of a mutant P protein lacking the recognized casein kinase II (CKII) sites suggested that hyperphosphorylation of VSV P protein was not due to a stimulation of CKII. We thus propose that expression of MxA in human brain cells is associated with the stimulation of a cellular kinase that is active in phosphorylating both cellular target proteins and VSV P protein.
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PMID:Expression of the human MxA protein is associated with hyperphosphorylation of VSV P protein in human neural Cells. 865 21

We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane.
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PMID:The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity. 889 94

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

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.
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PMID:In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein. 1072 Apr 65

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.
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PMID:Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses. 1660 81

Historical sources for the use of Glycyrrhiza species include ancient manuscripts from China, India and Greece. They all mention its use for symptoms of viral respiratory tract infections and hepatitis. Randomized controlled trials confirmed that the Glycyrrhiza glabra derived compound glycyrrhizin and its derivatives reduced hepatocellular damage in chronic hepatitis B and C. In hepatitis C virus-induced cirrhosis the risk of hepatocellular carcinoma was reduced. Animal studies demonstrated a reduction of mortality and viral activity in herpes simplex virus encephalitis and influenza A virus pneumonia. In vitro studies revealed antiviral activity against HIV-1, SARS related coronavirus, respiratory syncytial virus, arboviruses, vaccinia virus and vesicular stomatitis virus. Mechanisms for antiviral activity of Glycyrrhiza spp. include reduced transport to the membrane and sialylation of hepatitis B virus surface antigen, reduction of membrane fluidity leading to inhibition of fusion of the viral membrane of HIV-1 with the cell, induction of interferon gamma in T-cells, inhibition of phosphorylating enzymes in vesicular stomatitis virus infection and reduction of viral latency. Future research needs to explore the potency of compounds derived from licorice in prevention and treatment of influenza A virus pneumonia and as an adjuvant treatment in patients infected with HIV resistant to antiretroviral drugs.
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PMID:Antiviral effects of Glycyrrhiza species. 1788 24

It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations.
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PMID:X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells. 2037 2


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