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)

In a continuation of previous efforts to study the modified ATP requirements for RNA synthesis by poIR mutants of vesicular stomatitis virus (VSV), we have used a novel reconstitution assay to show that it is the template moiety of the mutants, not the polymerase proteins, which governs both the increased utilization of the ATP analog, beta, gamma-imido ATP (AMP-PMP), and the loss of a positive cooperativity-like response to varying ATP concentrations. Assays utilized uv-irradiated virus as a source of polymerase proteins and purified N-RNA as templates. Homologous and heterologous transcriptase reactions were carried out with wild-type (wt) virus and each of the two independently isolated poIR mutants. We show that in the presence of wt N-RNA template, substitution of AMP-PNP for ATP resulted in only approximately 5% of control RNA synthesis regardless of which source of polymerase was used. Furthermore, all reactions containing wt N-RNA template responded to varying ATP concentrations with a concave, upward-shaped Lineweaver-Burke plot generally indicative of positive cooperativity effects. In contrast, all reactions which utilized N-RNA templates from the poIR mutants showed an increased utilization of AMP-PNP (greater than 20%) and a more characteristic Michaelis-Menten response to changing ATP concentrations. These findings strongly support the notion that the template-associated nucleocapsid protein modulates the utilization of an ATP site which is directly or indirectly involved in VSV RNA synthesis.
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PMID:Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template. 284 22

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.
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PMID:Vesicular stomatitis virus transcription inhibited by purified MxA protein. 783 9

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