Coupled in vitro transcription and translation of vesicular stomatitis virus messenger RNA.

Breindl, Michael; Holland, John J.

doi:10.1073/pnas.72.7.2545

Cited by 25 publications

(13 citation statements)

References 29 publications

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“…Perhaps this protein is a nonglycosylated G protein precursor precipitated with anti-VSV IgG. This result is in agreement with previous data that there was little or no production of virus glycoproteins in cell-free translation systems (4,5,12).…”

Section: Discussionsupporting

confidence: 92%

In vitro translation of mRNA species from cells infected with Machupo virus

Lukashevich¹,

Stelmakh²,

Stchesljenok³

et al. 1987

Archives of Virology

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RNA from Machupo virus infected cells was centrifuged in a linear sucrose gradient and RNAs from gradient fractions were tested separately for template activity in a cell-free protein synthesizing system from rabbit reticulocytes. Fraction 15-16 S programmed the synthesis of protein that migrated in SDS-polyacrylamide gel like the nucleocapsid protein of a purified virus. The synthesis of virus glycoproteins was not detected in the system.

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Section: Discussionsupporting

confidence: 92%

In vitro translation of mRNA species from cells infected with Machupo virus

Lukashevich¹,

Stelmakh²,

Stchesljenok³

et al. 1987

Archives of Virology

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“…Incubation of the nucleocapsids in the presence of the four ribonucleoside triphosphates results in the synthesis of the leader RNA and all five viral messengers (2). To exclude that the in vitro synthesis of Gs protein was due to the presence of partially degraded G protein mRNA preparations, we performed a coupled transcription-translation experiment (6). VSV nucleocapsids were incubated in a reticulocyte lysate in the presence of dog pancreas microsomes, and the proteins synthesized were analyzed by SDS-PAGE (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The virus suspension was desalted by exclusion chromatography on Sephadex G-25. The viral nucleocapsids were prepared as described previously (6). VSV infection of cells.…”

Section: Methodsmentioning

confidence: 99%

The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process

Graeve¹,

Garreis-Wabnitz²,

Zauke³

et al. 1986

J Virol

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G, protein is a shorter, soluble form of the viral G protein of vesicular stomatitis virus (VSV) lacking the membrane-anchoring domain. Production of G, protein appears to be a general property of VSV because infection of BHK-21 cells by five different isolates of the VSV serotype Indiana led in all cases to the synthesis of Gs protein. Moreover, it is formed in a variety of eucaryotic cell lines after VSV infection. In pulse-chase experiments, we observed a time-dependent change in the ratio of G to Gs protein released into the growth medium, suggesting that Gs is formed intracellularly rather than on the cell surface. Further experiments revealed that G. protein can be synthesized in vitro in the reticulocyte lysate system after addition of a viral mRNA fraction and in a coupled transcription-translation system with VSV core particles. In the presence of microsomal membranes both G and Gs protein were glycosylated in the reticulocyte lysate, confirming that the authentic Gs protein is synthesized in vitro. The addition of various protease inhibitors to the cell-free system and variation of the incubation conditions did not alter the ratio of G to G. formation. Taken together, these experiments suggest strongly that Gs protein is not a product of a membrane-associated proteolytic activity but is formed during or shortly after the translation process. Our attempts to detect a specific, shorter mRNA coding for the G. protein by molecular hybridization procedures did not reveal the existence of such a mRNA species.

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“…The RNP complex was purified as described previously (6). Briefly, 1 mg of purified VSV was disrupted (0.4 M NaCl, 0.2% Triton X-100) for 1 h on ice.…”

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

124

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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Coupled in vitro transcription and translation of vesicular stomatitis virus messenger RNA.

Cited by 25 publications

References 29 publications

In vitro translation of mRNA species from cells infected with Machupo virus

In vitro translation of mRNA species from cells infected with Machupo virus

The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process

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

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