The Role of Polypeptides L and NS in the Transcription Process of Vesicular Stomatitis Virus New Jersey Using the Temperature-sensitive Mutant tsE1

Ongrádi, J; Cunningham, Charles; Szilágyi, J. F.

doi:10.1099/0022-1317-66-5-1011

Cited by 32 publications

(19 citation statements)

References 27 publications

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“…Nucleocapsid RNA (N RNA) template was purified from recombinant VSV essentially as described previously (31). Briefly, 4 mg purified virus was disrupted on ice for 1 h in 20 mM Tris-HCl (pH 8.0), 0.1% Triton X-100, 5% glycerol, 5 mM EDTA, 3.5 mM dithioerythritol, 20% dimethyl sulfoxide, and 1.0 M LiCl.…”

Section: Methodsmentioning

confidence: 99%

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

Lǐ

Rahmeh

Morelli

et al. 2008

J Virol

122

159

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Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5 monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5 diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.The 5Ј terminus of eukaryotic mRNA is modified by the addition of a 7 m GpppN cap structure. The cap structure is essential for mRNA stability, mRNA transportation, splicing of pre-mRNAs, and translation (13, 28). Formation of this structure requires a series of enzymatic reactions. An RNA triphosphatase (RTPase) hydrolyzes the 5Ј triphosphate (pppN) end of mRNA to yield a 5Ј diphosphate (ppN). This is capped by an RNA guanylyltransferase (GTase) which transfers Gp derived from GTP to form the cap structure. The cap is subsequently methylated by guanine-N-7 (G-N-7) methyltransferase (MTase) to yield 7 m GpppN, which can be further methylated by ribose-2Ј-O (2Ј-O) MTase to yield 7 m GpppN m (14, 36). Cap formation in nonsegmented negative-strand (NNS) RNA viruses involves a different reaction mechanism. For vesicular stomatitis virus (VSV) (2), spring viremia of carp virus (16), and respiratory syncytial virus (RSV) (5), the underlined phosphates of the 5Ј GpppN triphosphate bridge were shown to be derived from GDP rather than GMP. Recent studies with VSV demonstrated that this reaction does not involve transfer of guanylate onto the mRNA, but rather involves a polyribonucleotidyltransferase activity (29). Here, the mRNA capping reaction proceeds via a covalent intermediate between the 241-kDa viral polymerase protein L and the 5Ј monophosphate mRNA. This monophosphate mRNA is transferred onto GDP derived from GTP to yield the GpppA mRNA cap. Consequently, this mechanism of cap formation is in marked contrast with those catalyzed by conventional GTases. The crystal structures of representative GTases have been solved and their reaction mechanisms studied in biochemical detail (17). GTases typically contain a KxDG motif, in which the lysine forms the covalent intermediate with GMP, and consistent with...

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Section: Methodsmentioning

confidence: 99%

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

Lǐ

Rahmeh

Morelli

et al. 2008

J Virol

122

159

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“…Transcriptase activity was measured by the method of Szil/tgyi & Pringle (1975). During the incubation period 20 vtl samples were taken at intervals and spotted onto 2-5 cm discs of DE81 paper which were allowed to dry in air prior to washing with 5~ sodium pyrophosphate (five times), deionized water (twice) and ethanol (twice) (Ongr~di et al, 1985). After drying, toluene-based scintillant was added and radioactivity was counted in a Beckman scintillation counter for 1 min.…”

Section: Introductionmentioning

confidence: 99%

In vitro Transcriptase Deficiency of Temperature-dependent Host Range Mutants of Chandipura Virus

Rasool¹,

Pringle²

1986

Journal of General Virology

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SUMMARYTen of 12 Chandipura virus tdCE mutants, which exhibit temperature-dependent restriction of growth in chick embryo (CE) cells but not in BS-C-1 cells, showed deficient transcriptase activity in vitro at 39 °C relative to wild-type virus. A gradation in transcriptional activity at 39 °C in vitro was observed. Reversion of the tdCE phenotype to unrestricted growth in CE cells at 39 °C was accompanied by partial restoration of normal transcriptase activity at 39 °C, suggesting that reversion was mediated by either extragenic or intragenic suppression. Viral protein synthesis was reduced or absent in CE cells at 39 °C indicating that transcription was also defective in vivo under these conditions. Induction of heat-shock proteins in CE cells at 39 °C occurred normally in tdCE mutant-infected cells and RNA methylation in vitro was unaffected.

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“…According to the method of Ongradi et al [14), 500 pi of a fresh suspension of PI was further dissociated in a reaction mixture (4.5 ml) containing 60 mM Tris-HCl (pH 8.0), 10% glycerol. 3 mM DTT, I mM EDTA, O.l%digitonin.…”

Section: Dissociation O F Pi Into S2 and P2mentioning

confidence: 99%

In vitro Studies on the Nucleocapsid-Associated RNA Polymerase of Wheat Rosette Stunt Virus

Sun¹,

Gong²

1988

Intervirology

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RNA-dependent RNA polymerase activity was detected in both virion and nucleocapsid preparations of wheat rosette stunt virus, a plant rhabdovirus. The presence of nonionic detergent such as Nonidet P40 was essential for activity in reaction mixtures containing virions. The polymerase product was proved to be single-stranded RNA. By two-step controlled dissociation of the nucleocapsids, four subviral fractions (L protein, NS-N-RNA complex, NS protein, and N-RNA complex) were prepared. None of these fractions showed RNA polymerase activity when assayed individually. In experiments combining the various fractions, RNA synthesis was observed only when the L and NS proteins and the N-RNA complex were present in reaction mixtures.

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The Role of Polypeptides L and NS in the Transcription Process of Vesicular Stomatitis Virus New Jersey Using the Temperature-sensitive Mutant tsE1

Cited by 32 publications

References 27 publications

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

In vitro Transcriptase Deficiency of Temperature-dependent Host Range Mutants of Chandipura Virus

In vitro Studies on the Nucleocapsid-Associated RNA Polymerase of Wheat Rosette Stunt Virus

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