Synthesis of Bunyavirus-specific Proteins in a Continuous Cell Line (XTC-2) Derived from Xenopus laevis

Watret, Gillian E.; Pringle, C. R.; Elliott, Richard M.

doi:10.1099/0022-1317-66-3-473

Cited by 73 publications

(99 citation statements)

References 17 publications

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“…2a, lane 1), and this most probably represents L-N protein interaction in the cell extracts (which were not denatured prior to reaction with the antibodies), perhaps in the form of nucleocapsids. The antiserum prepared against purified Bunyamwera virus (Watret et al, 1985) immunoprecipitated L from Bunyamwera virus-infected cells but not from recombinant vaccinia virus-infected cells that express the L protein (H. Jin & R. M. Elliott, unpublished observations). Thus in this case we suggest that L is precipitated because it is associated with the N protein which is the major antigenic target.…”

Section: Discussionmentioning

confidence: 99%

“…Bunyamwera virus was grown in BHK cells as described previously (Watret et al, 1985). Recombinant vaccinia viruses vTF7-3 (which expresses bacteriophage T7 RNA polymerase; Fuerst et al, 1986), and vSCll-BUNL and vTF7-5BUNL (which 0001-1044 © 1992 SGM contain the Bunyamwera virus L gene cDNA; Jin & Elliott, 1991) were grown in CV-I ceils as described by Mackett et al (1985).…”

Section: Methodsmentioning

confidence: 99%

“…Radiolabelling of infected cells with [3sS]methionine, preparation of cell lysates, formation of immune complexes and analysis of proteins by PAGE were as described in Watret et al (1985). Five p.l of anti-fusion protein sera was used in each reaction.…”

Section: Characterization Of" Antiseramentioning

confidence: 99%

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Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Jin

Elliott

1992

Journal of General Virology

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Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino-or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Jin

Elliott

1992

Journal of General Virology

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“…The attenuated MP12 strain of Rift Valley fever virus (RVFV) was kindly provided by Michele Bouloy, Institut Pasteur, Paris, France (13). BUNV was kindly provided from Richard M. Elliott, Institute of Virology, University of Glasgow, U.K. (14).…”

Section: Methodsmentioning

confidence: 99%

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

Kochs

Janzen

Hohenberg

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

191

170

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“…RNAs were translated in a rabbit reticuloyte lysate (Amersham) using 10 ~tg total RNA in a 25 ~tl reaction containing 50 ktCi [35S]methionine. Radiolabelled translation products were separated on SDS-polyacrylamide gels (acrylamide : bisacrylamide 73 "1 ; Watret et al, 1985) using the discontinuous buffer system of Laemmli.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Mutations Responsible for the Electrophoretic Mobility Differences in the NS Proteins of Vesicular Stomatitis Virus New Jersey Complementation Group E Mutants

Rae¹,

Elliott²

1986

Journal of General Virology

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SUMMARYTemperature-sensitive (ts) mutants of vesicular stomatitis virus, New Jersey serotype, classified in complementation group E contain lesions in the NS gene, which manifest as marked electrophoretic mobility differences of the mutant NS proteins in SDS-polyacrylamide gels. We have cloned full-length cDNA copies of the mutant NS mRNAs, and have determined their nucleotide sequences, tsE1 and tsE3 had single nucleotide changes, and tsE2 had two nucleotide changes, compared to the wild-type NS gene.

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Synthesis of Bunyavirus-specific Proteins in a Continuous Cell Line (XTC-2) Derived from Xenopus laevis

Cited by 73 publications

References 17 publications

Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

Characterization of the Mutations Responsible for the Electrophoretic Mobility Differences in the NS Proteins of Vesicular Stomatitis Virus New Jersey Complementation Group E Mutants

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