Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Mar;71(3):2264–2269. doi: 10.1128/jvi.71.3.2264-2269.1997

Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus.

M M Goodin 1, B Schlagnhaufer 1, T Weir 1, C P Romaine 1
PMCID: PMC191334  PMID: 9032361

Abstract

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  2. Ben-Tzvi B. S., Koltin Y., Mevarech M., Tamarkin A. RNA polymerase activity in virions from Ustilago maydis. Mol Cell Biol. 1984 Jan;4(1):188–194. doi: 10.1128/mcb.4.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buck K. W., Ratti G. Molecular weight of double-stranded RNA: a re-examination of Aspergillus foetidus virus S RNA components. J Gen Virol. 1977 Oct;37(1):215–219. doi: 10.1099/0022-1317-37-1-215. [DOI] [PubMed] [Google Scholar]
  4. Buck K. W. Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase. Biochem Biophys Res Commun. 1978 Oct 16;84(3):639–645. doi: 10.1016/0006-291x(78)90753-2. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  7. Fujimura T., Wickner R. B. Reconstitution of template-dependent in vitro transcriptase activity of a yeast double-stranded RNA virus. J Biol Chem. 1989 Jun 25;264(18):10872–10877. [PubMed] [Google Scholar]
  8. Fujimura T., Wickner R. B. Replicase of L-A virus-like particles of Saccharomyces cerevisiae. In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form. J Biol Chem. 1988 Jan 5;263(1):454–460. [PubMed] [Google Scholar]
  9. Ghabrial S. A. New developments in fungal virology. Adv Virus Res. 1994;43:303–388. doi: 10.1016/S0065-3527(08)60052-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  11. Harmsen M. C., Tolner B., Kram A., Go S. J., de Haan A., Wessels J. G. Sequences of three dsRNAs associated with La France disease of the cultivated mushroom (Agaricus bisporus). Curr Genet. 1991 Jul;20(1-2):137–144. doi: 10.1007/BF00312776. [DOI] [PubMed] [Google Scholar]
  12. Matsumoto Y., Fishel R., Wickner R. B. Circular single-stranded RNA replicon in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7628–7632. doi: 10.1073/pnas.87.19.7628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Romaine C. P., Schlagnhaufer B., Goodin M. M. Vesicle-associated double-stranded ribonucleic acid genetic elements in Agaricus bisporus. Curr Genet. 1994 Feb;25(2):128–134. doi: 10.1007/BF00309538. [DOI] [PubMed] [Google Scholar]
  14. Romaine C. P., Schlagnhaufer B. PCR analysis of the viral complex associated with La France disease of Agaricus bisporus. Appl Environ Microbiol. 1995 Jun;61(6):2322–2325. doi: 10.1128/aem.61.6.2322-2325.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  16. Sclafani R. A., Fangman W. L. Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands. Mol Cell Biol. 1984 Aug;4(8):1618–1626. doi: 10.1128/mcb.4.8.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sriskantha A., Wach M. P., Schlagnhaufer B., Romaine C. P. Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus. J Virol. 1986 Mar;57(3):1004–1009. doi: 10.1128/jvi.57.3.1004-1009.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. White T. C., Wang C. C. RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia. Nucleic Acids Res. 1990 Feb 11;18(3):553–559. doi: 10.1093/nar/18.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. van der Lende T. R., Harmsen M. C., Wessels J. G. Double-stranded RNAs and proteins associated with the 34 nm virus particles of the cultivated mushroom Agaricus bisporus. J Gen Virol. 1994 Sep;75(Pt 9):2533–2536. doi: 10.1099/0022-1317-75-9-2533. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES