1887

Abstract

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.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-73-9-2235
1992-09-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/73/9/JV0730092235.html?itemId=/content/journal/jgv/10.1099/0022-1317-73-9-2235&mimeType=html&fmt=ahah

References

  1. Antic D., Lim B.-U., Kang C. Y. 1991; Nucleotide sequence and coding capacity of the large (L) genomic RNA segment of Seoul 8039 virus, a member of the hantavirus genus. Virus Research 19:59–66
    [Google Scholar]
  2. Boyer P. L., Ferris A. L., Hughes S. H. 1992; Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. Journal of Virology 66:1031–1039
    [Google Scholar]
  3. Bressan G. M., Stanley K. K. 1987; pUEX, a bacterial expression vector related to pEX with universal host specificity. Nucleic Acids Research 15:10056
    [Google Scholar]
  4. de Haan P., Kormelink R., Resende R., de O., van Poelwijk F., Peters D., Goldbach R. 1991; Tomato spotted wilt virus L RNA encodes a putative RNA polymerase. Journal of General Virology 72:2207–2216
    [Google Scholar]
  5. Delarue M., Poch O., Tordo N., Moras D., Argos P. 1990; An attempt to unify the structure of polymerases. Protein Engineering 3:461–467
    [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
    [Google Scholar]
  7. Elliott R. M. 1989; Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae. Virology 173:426–436
    [Google Scholar]
  8. Elliott ., M R. 1990; Molecular biology of the Bunyaviridae. Journal of General Virology 71:501–522
    [Google Scholar]
  9. Elliott R. M., Dunn E., Simons J. F., Pettersson R. F. 1992; Nucleotide sequence and coding strategy of the Uukuniemi virus L RNA segment. Journal of General Virology 73:1745–1752
    [Google Scholar]
  10. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesises bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, U.S.A. 83:8122–8126
    [Google Scholar]
  11. Inokuchi V., Hirashima A. 1987; Interference with viral infection by defective RNA replicase. Journal of Virology 61:3946–3949
    [Google Scholar]
  12. Jablonski S. A., Luo M., Morrow C. D. 1991; Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. Journal of Virology 65:4565–4573
    [Google Scholar]
  13. Jin H. 1991; Expression of the Bunyamwera virus polymerase gene. Ph,. D. thesis, University of Glasgow;
    [Google Scholar]
  14. Jin H., Elliott R. M. 1991; Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses. Journal of Virology 65:4182–4189
    [Google Scholar]
  15. Kolakofsky D., Hacker D. 1991; Bunyavirus RNA synthesis: genome transcription and replication. In Bunyaviridae pp 143–159 Edited by Kolakofsky D. Current Topics in Microbiology and Immunology, vol 169 Berlin: Springer-Verlag;
    [Google Scholar]
  16. Mackett M., Smith G. L., Moss B. 1985; The construction and characterization of vaccinia virus recombinants expressing foreign genes. In DNA Cloning vol 2 pp 191–211 Edited by Rickwood D., Hames B. D. Oxford: IRL Press;
    [Google Scholar]
  17. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  18. Muller R., Argentina C., Bouloy M., Prehaud C., Bishop D. H. L. 1991; Completion of the genome sequence of Rift Valley fever phlebovirus indicates that the L RNA is negative sense or ambisense and codes for a putative transcriptase-replicase. Nucleic Acids Research 19:5433
    [Google Scholar]
  19. Nakamaye K. L., Eckstein F. 1986; Inhibition of restriction endonuclease cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Research 14:9670–9698
    [Google Scholar]
  20. Obijeski J. F., Bishop D. H. L., Palmer E., Murphy F. A. 1976; Segmented genome and nucleocapsid of La Crosse virus. Journal of Virology 20:664–675
    [Google Scholar]
  21. Pattnaik A. K., Wertz G. W. 1990; Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. Journal of Virology 64:2948–2957
    [Google Scholar]
  22. Pattnaik A. K., Wertz G. W. 1991; Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly and budding of defective interfering particles. Proceedings of the National Academy of Sciences, U.S.A. 88:1379–1383
    [Google Scholar]
  23. Poch O., Sauvaget I., Delarue M., Tordo N. 1989; Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO Journal 8:3867–3875
    [Google Scholar]
  24. Schmaljohn C. S. 1990; Nucleotide sequence of the L genome segment of Hantaan virus. Nucleic Acids Research 18:6728
    [Google Scholar]
  25. Smith A. R., Boursnell M. E. G., Binns M. M., Brown T. D. K., Inglis S. C. 1990; Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus. Journal of General Virology 71:3–11
    [Google Scholar]
  26. Stohwasser R., Raab K., Darai G., Bautz E. K. F. 1991; Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase. Virology 183:386–391
    [Google Scholar]
  27. Studier F. M., Rosenberg A., Dunn J. J. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185:60–89
    [Google Scholar]
  28. Vieira J., Messing J. 1987; Production of single-stranded DNA. Methods in Enzymology 153:3–11
    [Google Scholar]
  29. Watret G. E., Pringle C. R., Elliott R. M. 1985; Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopus laevis . Journal of General Virology 66:473–482
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-73-9-2235
Loading
/content/journal/jgv/10.1099/0022-1317-73-9-2235
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error