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 addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.
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PMID:Two methyltransferase activities in the purified virions of vesicular stomatitis virus. 20 77

We have previously shown that the vesicular stomatitis virus (VSV) host range mutant, hr 1, is completely defective for the mRNA methyltransferase activities, but can synthesize full-length, unmethylated mRNAs in vitro [S. M. Horikami and S. A. Moyer (1982). Proc. Natl. Acad. Sci. USA 79, 7694-7698] and in vivo [S. M. Horikami, F. De Ferra, and S. A. Moyer (1984). Virology 138, 1-15]. Here we have used the hr 1 mutant to identify the viral protein which possesses the methyltransferase activities. The wild-type VSV L and NS proteins, subunits of the viral RNA polymerase, were separately purified and added to high salt dissociated mutant hr 1 nucleocapsids for in vitro transcription reactions. The results show that the purified wild-type L protein, but not the NS protein, restores methylation and thus possesses the viral mRNA methyltransferase activities.
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PMID:The vesicular stomatitis virus L protein possesses the mRNA methyltransferase activities. 283 58

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.
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PMID:Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA. 618 92

The viral RNA polymerase of detergent-treated vesicular stomatitis virus normally synthesizes viral mRNAs in vitro that are both guanylylated and methylated to give 5'-terminal 7mGpppAm caps. We have characterized a virus host range mutant, hr 1, that is totally defective in vitro in the methylation of mRNA, although full-length polyadenylylated mRNAs with 5' termini of the form GpppA are synthesized in normal yields. A second mutant, hr 8, is partially defective in methylation and synthesizes mRNAs in vitro with primarily GpppA and some GpppAm 5' termini. When used for in vitro translation, the unmethylated hr 1 mutant mRNA shows, as expected, reduced synthesis of viral proteins. These data provide direct evidence that the vesicular stomatitis virus-associated methyltransferase activities are virus encoded.
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PMID:Host range mutants of vesicular stomatitis virus defective in in vitro RNA methylation. 629 46

Lentiviral vectors may improve hematopoietic stem cell (HSC) gene transfer because of their enhanced ability to transduce nondividing cells. However, many studies report efficient transduction only at high multiplicities of infection (MOI). This study reports efficient transduction of human CD34(+) cells with a drug resistance gene allowing post-transduction selection using lentivirus under low-MOI conditions that did not require cytokine stimulation or viral concentration. We used the P140K methylguanine-DNA-methyltransferase mutant (P140K MGMT) as the gene insert into a second-generation lentiviral backbone and triple-plasmid transfection to generate vesicular stomatitis virus (VSV)-G protein-pseudotyped virus. The P140K MGMT gene product, O(6)-alkylguanine-DNA-alkyltransferase (AGT), provides protection from the therapeutic drug combination of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the wild-type AGT inhibitor O(6)-benzylguanine (BG). Low-speed spinoculation enhanced transduction more than addition of Polybrene or multiple virus exposures. Addition of cytokines was not required. Low-MOI transduction (< or =1) of human CD34(+) and CD34(+) lin(-) cells with P140K MGMT lentivirus resulted in an average 41% and 89% gene transfer rate as assessed by PCR, respectively, and concordant AGT expression that conferred substantial clonogenic survival advantage after BG/BCNU treatment. During in vitro drug selection, 87% of surviving CD34(+) cell-derived colony-forming units (CFU) were transduced. This work shows the potential utility of lentiviral vectors for drug resistance gene transfer to HSCs for the purpose of in vivo selection and marrow protection. Because drug selection will enrich for transduced progenitors, high MOI can be avoided, improving the safety profile of lentiviral gene transfer.
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PMID:Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. 1194 64

The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5'-cap structures methylated at the guanine-N7 and 2'-O-adenosine positions (7mGpppA(m)). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34 degrees C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40 degrees C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5'-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.
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PMID:A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. 1591 87

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.
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PMID:Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. 1622 59

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.
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PMID:Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation. 1653 83

There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3' end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5' cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM. To investigate this unusual phenotype, we assayed the effects of SAH on transcription using a panel of recombinant viruses that contained mutations in domain VI of the VSV L protein. The L proteins we investigated displayed a range of 5' cap methyltransferase activities. In the present study, we show that the ability of the VSV L protein to catalyze methyl transfer correlates with its sensitivity to SAH with respect to polyadenylation, thereby indicating an intriguing connection between 5' and 3' end mRNA modifications. We also identified an L protein mutant that hyperpolyadenylates mRNA irrespective of the presence or absence of exogenous SAH. Further, the data presented here show that the wild-type L protein hyperpolyadenylates a percentage of VSV mRNAs in infected cells as well as in vitro.
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PMID:S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein. 1882 53

Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.
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PMID:Identification of aromatic amino acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus is essential for both guanine-N-7 and ribose 2'-O methyltransferases. 2096 92


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