UMLS:C0038362 - FACTA Search

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Query: UMLS:C0038362 (stomatitis)
8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soluble transcriptase containing the L and the NS proteins was isolated from purified vesicular stomatitis virus and its binding with the template ribonucleoprotein containing the N protein-RNA complex was studied with respect to its ability to initiate and synthesize RNA in vitro. By using u.v.-irradiated template reconstituted with soluble transcriptase, it was shown that the synthesis of leader RNA and other small initiated mRNA sequences continued while full-length mRNA synthesis decreased by 90%. In the presence of ATP and CTP, the reconstituted complex synthesized polyphosphorylated oligonucleotides which include AC, AAC and AACA which represent 5'-terminal sequences transcribed from the leader template and genes coding for mRNAs. In the presence of arabinosyl ATP, an inhibitor of RNA synthesis in vitro, the synthesis of leader RNA was found to be inhibited considerably more than other small initiated mRNA sequences. Reconstitution of RNA synthesis with soluble transcriptase and template in the presence of viral matrix (M) protein at low ionic condition resulted in virtual cessation of leader RNA synthesis, although the synthesis of small initiated N mRNA, 11 to 14 bases, continued. These results suggest that transcriptase can bind at multiple sites on the genome template and initiate RNA chains.
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PMID:Interaction of L and NS proteins of vesicular stomatitis virus with its template ribonucleoprotein during RNA synthesis in vitro. 619 59

The objective of this investigation was to examine by electron microscopy the replicative ribonucleoprotein (RNP) structures synthesized in vesicular stomatitis virus-infected HeLa cells. Pulse-labeled in vivo products of vesicular stomatitis virus replication and transcription can be separated by centrifugation in Renografin gradients. Transcription complexes are dissociated, allowing nascent messenger RNPs to remain at the top of the gradient, whereas RNPs biochemically consistent with replication complexes sediment to the middle of the gradient. Examination of these structures by electron microscopy revealed that all exist as coiled or helical RNPs having dimensions of approximately 20 by 700 nm. These structures can be further subdivided into three major morphological classes: (i) linear forms (20 by 769 +/- 158 nm), which have both ends free; (ii) circular forms (20 by 679 +/- 95 nm), which appear to have both ends joined; and (iii) complex forms, which include those structures which are branched replicative complexes as well as those which are random. To distinguish random complexes and possible transcriptive complex contaminants from replicative complexes, it was necessary to uncoil the RNP structures with EDTA so that length measurements could be made relating the nascent strand length to its position on the template. After EDTA treatment, the linear RNPs uncoiled (10 by 4,035 +/- 3,802 nm), and the circular morphology virtually disappeared. However, a new form appeared which was one-half the length and double the width (20 by 2,103 +/- 306 nm) of full-length RNPs and contained a loop at one end and two free ends at the other (alpha-form RNP). The distribution and length analysis of these structures, plus and minus EDTA, suggest that the alpha-form RNPs arise by EDTA-induced uncoiling of circular forms held together at the ends. Close scrutiny of uncoiled complex RNPs revealed no single-strand RNP templates with single-strand nascents. However, several complexes were observed which appeared to contain alpha-form templates with single-strand nascent RNPs. Length measurements suggest these complexes are neither random nor transcriptive, but are replicative. These experiments suggest that replication may, in part, occur on circular coiled RNP templates.
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PMID:Electron microscopy of vesicular stomatitis virus replicative ribonucleoproteins. 624 10

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.
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PMID:Translational control of protein synthesis after infection by vesicular stomatitis virus. 625 23

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, "P." We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.
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PMID:Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus? 626 24

An enzyme activity that synthesizes cytidylyl (5'-3') guanosine 5'-triphosphate (pppGpC) in vitro has been identified in purified vesicular stomatitis virus. The activity is discernible after a lag period which is reduced in length with increasing virus concentration. The lag is eliminated by addition of pppGpC or ppGpC which are effective primers and stimulate dinucleotide synthesis linearly. The requirements of the reaction with respect to MgCl2, NaCl, and temperature are similar to those for viral mRNA synthesis in vitro. The activity, together with the viral L and NS proteins, is removed from virions by treatment with 0.8 M NaCl. The particulate fraction from infected cells that contains the transcribing subviral ribonucleoprotein particles also contains the enzyme activity. The corresponding fraction from uninfected cells does not, indicating that the activity is mediated by virus-specific proteins. Possible functions of the dinucleotide in the life cycle of the virus are discussed.
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PMID:Purified vesicular stomatitis virus contains an enzyme activity that synthesizes cytidylyl (5'-3') guanosine 5'-triphosphate in vitro. 627 45

An mRNA-ribonucleoprotein particle (mRNP) was found in vesicular stomatitis virus (VSV)-infected Chinese hamster ovary cells. The particle was present 3 and 4.5 h after infection but was barely discernible at 2 h. The mRNP (buoyant density, 1.56 g/cm3), which cosedimented with viral nucleocapsid in a sucrose density gradient at approximately 120 to 160S, was separable from nucleocapsid (buoyant density, 1.31 g/cm3) by CsCl density gradient centrifugation. It contained all five VSV mRNAs and, almost exclusively, viral N protein. Some host mRNA and host protein was also present in the particle. The intact mRNP was incapable of stimulating protein synthesis in an in vitro protein-synthesizing system, although the VSV mRNA isolated from the particle by phenol extraction was functional in vitro. In contrast, intact polysomes stimulated cell-free protein synthesis to the same extent as purified polysomal mRNA. By 4.5 h after infection, 97% of the functional mRNA in vivo was associated with the mRNP, and only 3% was on polysomes. The amount of polysomal mRNA at 4.5 h after infection was only 31% of that found at 2 h after infection; this was reflected by the 76% decrease observed in the rate of in vivo protein synthesis at 4.5 h relative to that found at 2 h. Thus, it appears that the mRNP serves as an organelle which sequesters the large excess of VSV mRNA that is normally made during secondary transcription.
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PMID:Translational control of vesicular stomatitis virus protein synthesis: isolation of an mRNA-sequestering particle. 629 40

Synthesis of a small amount of 42S RNA in addition to the VSV specific mRNA species was observed in a coupled transcription-translation system containing ribonucleoprotein particles from L cell infected with vesicular stomatitis virus and nuclease-treated ribosomal extract obtained from uninfected HeLa cells. Analysis on a CsCl density gradient showed that the synthesized 42S RNA was associated with newly synthesized by protein as a nucleoprotein of bouyant density of 1.3 g/ml. The 42S RNA and the N protein present in the nucleoprotein were resistant to nuclease and protease, respectively. About 35% of the remaining 65% had a complementary polarity. The evidence presented here demonstrates that both the full length genomic and the complementary RNA are associated with N protein in the in vitro replication process. A template role for the complementary 42S RNA for replication of the genomic RNA is also suggested.
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PMID:Synthesis in vitro of full length genomic RNA and assembly of the nucleocapsid of vesicular stomatitis virus in a coupled transcription-translation system. 629

The effect of proteins soluble in acidic chloroform-methanol (ACMS proteins) on the transcriptase activity of virus ribonucleoproteins (RNPs) in vitro has been studied. Experiments with ACMS membrane (M) proteins from type A and B orthomyxoviruses, as well as from vesicular stomatitis virus, showed that inhibition of the viral RNP transcriptase activity occurred when they interacted with M proteins isolated from viruses of a different serotype, or even of a different family. The presence of ACMS proteins capable of inhibiting the transcriptase activity of orthomyxovirus RNP in vitro was also detected in human blood plasma and among proteins produced by human leukocytes. Determination of the minimum concentration of M protein inhibiting the RNP transcriptase activity, and analysis of the fowl plague virus M protein-RNP complex formed in the in vitro system, showed that the M protein was capable of inhibiting RNP transcriptase activity at a M:RNP ratio of 0.1 to 0.2:1.
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PMID:In vitro inhibition of negative strand virus transcriptase activity by proteins soluble in acidic chloroform-methanol. 630 Feb 85

Small leader RNAs, copied from the extreme 3' ends of the minus and plus strands of the vesicular stomatitis virus (VSV) genome, are thought to play a central role in the regulation of viral transcription and replication. We describe here a novel class of VSV mutants, denoted pol R, in which termination at leader sites in vitro is specifically suppressed. We have assayed for the presence of leader RNAs and readthrough transcripts in reaction products from standard virion templates (plus leader) and defective interfering particle templates (minus leader). In both cases, mutant virions gave rise to a much higher proportion of readthrough transcripts than wild type (greater than 80% vs approximately 10%). Reconstitution experiments with separated ribonucleoprotein (RNP) templates and polymerase protein fractions revealed, surprisingly, that the N protein moiety of the RNP template was responsible for readthrough. This conclusion was further supported by protein analyses that showed a similar charge change in the N protein of two independently isolated pol R VSV mutants. These results lead us to propose that modification of the N protein may regulate termination at leader RNA sites.
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PMID:RNP template of vesicular stomatitis virus regulates transcription and replication functions. 631 23

Microinjection of purified transcriptionally active ribonucleoprotein (RNP) complex of vesicular stomatitis virus in vero cells resulted in the production and release of virus. Compared to the release of virus by cells treated with RNP in the presence of DEAE-dextran, the microinjection technique was highly efficient. Microinjection in Xenopus oocytes also resulted in initiation of infection as shown by the synthesis of virus-specific proteins in the cell cytoplasm. It was further observed that RNP stripped of L protein but containing residual NS protein was capable of initiating virus production or protein synthesis when microinjected in vero cells or in oocytes, respectively. Since L and NS proteins are essential for in vitro transcription by RNP, these results suggest that a trace amount of L protein may remain bound to the RNP and a host factor may stimulate residual L activity in vivo.
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PMID:Microinjection of vesicular stomatitis virus ribonucleoprotein into animal cells yields infectious virus. 631 70

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